Nucleic acid module coding for αglucosidase, plants that synthesize modified starch, methods for the production and use of said plants, and modified starch

ABSTRACT

The present invention relates to nucleic acid molecules which encode a protein with the activity of a potato α-glucosidase and to processes for the generation of transgenic plant cells and plants which synthesize a modified starch. Moreover, the present invention relates to vectors and host cells comprising the nucleic acid molecules according to the invention, to the plant cells and plants originating from the processes according to the invention, to the starch synthesized by the plant cells and plants according to the invention, and to processes for the production of this starch.

The present invention relates to nucleic acid molecules which encode aprotein with the activity of a potato α-glucosidase, and to processesfor the generation of transgenic plant cells and plants which synthesizea modified starch. Moreover, the present invention relates to vectorsand host cells comprising the nucleic acid molecules according to theinvention, to the plant cells and plants originating from the processesaccording to the invention, to the starch synthesized by the plant cellsand plants according to the invention, and to processes for theproduction of this starch.

Taking into consideration the increasing importance attached to plantconstituents as renewable raw materials, biotechnology research attemptsto adapt plant-based raw materials to the needs of the processingindustry. To allow renewable raw materials to be used in as many fieldsof application as possible, it is therefore necessary to provide amultiplicity of substances.

Besides oils, fats and proteins, polysaccharides constitute importantrenewable raw materials from plants. Besides cellulose, starch, which isone of the most important storage substances in higher plants, occupiesa central position amongst the polysaccharides. Besides maize, rice andwheat, potatoes play an important role, in particular in starchproduction.

The polysaccharide starch is a polymer of chemically uniform units, theglucose molecules. However, it is a highly complex mixture of differentforms of molecules which differ with regard to their degree ofpolymerization and the occurrence of branchings of the glucose chains.Starch therefore constitutes no uniform raw material. In particular, wedifferentiate between amylose starch, an essentially unbranched polymerof α-1,4-glycosidically linked glucose molecules, and amylopectinstarch, which, in turn, constitutes a complex mixture of differentlybranched glucose chains. The branchings are generated by the occurrenceof additional α-1,6-glycosidic linkages. In typical plants used forstarch production such as, for example, maize or potatoes, the starchsynthesized consists of approx. 25% amylose starch and approx. 75%amylopectin starch.

The molecular structure of starch, which is determined to a great extentby the degree of branching, the amylose/amylopectin ratio, the averagelength and distribution of the side chains, and the presence ofphosphate groups, is decisive for important functional properties ofstarch or its aqueous solutions. Examples of functional properties whichmust be mentioned in this context are solubility, the retrogradationbehavior, the film-forming properties, the viscosity—the colorstability, the gelatinization properties, and binding and adhesiveproperties. The starch granule size may also be of importance forvarious uses. Also, the generation of high-amylose starches is ofparticular interest for certain applications. Furthermore, a modifiedstarch present in plant cells can advantageously modify the behavior ofthe plant cell under certain conditions. For example, it is feasible toreduce starch breakdown during the storage of starch-containing organs,such as, for example, seeds or tubers, prior to their furtherprocessing, for example for extracting the starch. It is furthermore ofinterest to prepared modified starches which lead to plant cells orplant organs containing this starch being better suited to processing,for example in the production of foods such as popcorn or cornflakesfrom maize, or the production of French fries, chips or potato powderfrom potatoes. Of particular interest in this context is an improvementof the starches with regard to reduced cold sweetening, i.e. a reducedliberation of reducing sugars (in particular glucose) upon prolongedstorage at low temperatures. Potatoes especially are frequently storedat temperatures from 4 to 8° C. in order to minimize starch breakdownduring storage. The reducing sugars liberated during this process, inparticular glucose, result in undesired browning reactions (so-calledMaillard reactions) in the production of French fries or crisps.

The starch which can be isolated from plants is frequently adapted toparticular industrial purposes with the aid of chemical modificationswhich, as a rule, require time and money. It seems therefore desirableto find possibilities of generating plants which synthesize starch whoseproperties already meet the specific demands of the processing industryand thus combine economical and ecological advantages.

One possibility of providing such plants is, in addition to plantbreeding measures, the directed genetic modification of the starchmetabolism of starch-producing plants by recombinant methods. However, aprerequisite therefor is the identification and characterization of theenzymes which participate in starch synthesis modificaton and starchbreakdown (starch metabolism) and the isolation of the corresponding DNAsequences which encode these enzymes.

The biochemical synthetic pathways which lead to the synthesis of starchare essentially known. In plant cells, starch synthesis takes place inthe plastids. In photosynthetically active tissues, these plastids arethe chloroplasts, in photosynthetically inactive, starch-storing tissuethe amyloplasts.

Important enzymes which are involved in starch metabolism are, forexample, the branching enzymes, ADP glucose pyrophosphorylases,granule-bound starch syntheses, soluble starch synthases, debranchingenzymes, disproportioning enzymes, plastid starch phosphorylases, the R1enzymes (R1 proteins), amylases or glucosidases.

It is an object of the present invention to provide other, oralternative, recombinant approaches for modifying the starch metabolismin starch-synthesizing plants (for example rye, barley, oats, maize,wheat, sorghum and millet, sago, rice, peas, marrowfat peas, cassava,potatoes, tomatoes, oilseed rape, soybeans, hemp, flax, sunflowers,cowpeas, mung beans, beans, bananas or arrowroot or) suitable nucleicacid molecules by means of which plant cells can be transformed, thusallowing the synthesis of modified, advantageous starch species.

Such modified starch species exhibit, for example, modificationsregarding their degree of branching, the amylose/amylopectin ratio, thephosphate content, the starch granule size and/or the average length anddistribution of the side chains (i.e. side chain structure).

It is a further object of the invention to provide methods which allowthe generation of transgenic plants which synthesize a modified starchspecies.

Surprisingly, transgenic plants which have been transformed with thenucleic acid molecules according to the invention synthesize a starchwhose physicochemical properties and/or whose side chain structure ismodified in the particular manner so that the abovementioned objects areachieved by providing the use forms specified in the claims.

The invention therefore relates to a nucleic acid molecule encoding aprotein with the function of a potato α-glucosidase, selected from thegroup consisting of

a) nucleic acid molecules which encode a protein which encompasses theamino acid sequence stated under SEQ ID NO: 2 or its derivatives orparts,

b) nucleic acid molecules which encompass the nucleotide sequence shownunder SEQ ID NO: 1 or its derivatives or parts, or a correspondingribonucleotide sequence;

c) nucleic acid molecules which hybridize with, or are complementary to,preferably which hybridize specifically with, the nucleic acid moleculesstated under a) or b), and

d) nucleic acid molecules whose nucleotide sequence deviates from thesequence of the nucleic acid molecules stated under a), b) or c) owingto the degeneracy of the genetic code.

Accordingly, the present invention relates to a nucleic acid moleculewhich encodes an α-glucosidase and which comprises an amino acidsequence of SEQ ID NO: 2 or its derivatives or parts in accordance withthe cDNA insert of the plasmid (DSM No. 12347). The abovementionedα-glucosidase according to the invention is involved in the starchmetabolism of potatoes and is directly or indirectly involved in starchbiosynthesis.

The term “derivative” with regard to the α-glucosidase protein (or itspolypeptide, amino acid sequence) of the invention encompasses, for thepurposes of the present invention, a polypeptide which is derived fromSEQ ID NO: 2 and which comprises at least 163 amino acid residues,preferably at least 227, in particular at least 293 and very especiallypreferably approximately 309-322 amino acid residues which are selectedfrom the group of the amino acid residues consisting of

18H, 25 R, 34 G, 37 H, 38 G, 39 V, 41 L, 42 L, 44 S, 45 N, 46 G, 47 M,48 D, 51 Y, 53 G, 55 R, 56 I, 58 Y, 60 V, 61 I, 62 G, 63 G, 65 I, 66 D,67 L, 68 Y, 70 F, 71 A, 72 G, 75 P, 78 V, 81 Q, 83 T, 86 I, 87 G, 86 R,89 P, 90 A, 92 M, 93 P, 94 Y, 95 W, 97 F, 98 G, 99 F, 101 Q, 102 C,103R, 105G, 106 Y, 115 V, 116 V, 119 Y, 120 A, 124 I, 125 P, 126 L, 127E, 128 V, 129 M, 130 W, 131 T, 132 D, 133 I, 134 D, 135 Y, 136 M, 137 D,140 K, 141 D, 142 F, 143 T, 144 L, 145 D, 146 P, 147 V, 149 F, 150 P,157 F, 161 L, 162 H, 164 N, 166 Q, 168 Y, 169 V, 171 I, 173 D, 174 P,175 G, 176 I, 182 Y, 184 T, 187 R, 188 G, 189 M, 193 V, 194 F 196 K, 197R, 201 P, 202 Y, 204 G, 206 V, 207 W, 208 P, 209 G, 211 V, 212 Y, 214 P,215 D, 216 F, 217 L, 219 P, 224 F, 225 W, 228 E, 229 I, 232 F, 237 P,239 D, 240 G, 242 W, 244 D, 245 M, 246 N, 247 E, 249 S, 250 N, 251 F,252 I, 254 S, 260 S, 263 D, 265 P, 266 P, 267 Y, 268 K, 269 I, 270 N,271 N, 272 S, 273 G, 277 P, 278 I, 282 T, 284 P, 286 T, 289 H, 291 G,295 E, 296 Y, 299 H, 300 N, 301 L, 303 G, 305 L, 306 E, 310 T, 313 A,322 P, 323 F, 325 L, 327 R, 328 S, 329 T, 330 F, 333 S, 334 G, 336 Y,337 T, 339 H, 340 W, 341 T, 342 G, 343 D, 344 N, 345 A, 346 A, 348 W,350 D, 351 L, 353 Y, 354 S, 355 I, 356 P, 359 L, 361 F, 362 G, 363 L,364 F, 365 G, 367 P, 368 M, 370 G, 371 A, 372 D, 373 I, 374 C, 375 G,376 F, 380 T, 381 T, 382 E, 383 E, 384 L, 385 C, 387 R, 388 W, 389 I,390 Q, 391 L, 392 G, 393 A, 394 F, 395 Y, 396 P, 397 F, 399 R, 400 D,401 H, 402 S, 406 T, 409 Q, 410 E, 411 L, 412 Y, 414 W, 416 S, 417 V,418 A, 421 A, 424 V, 425 L, 426 G, 427 L, 428 R, 431 L, 432 L, 433 P,436 Y, 438 L, 439 M, 440 Y, 442 A, 446 G, 448 P, 449 I, 450 A, 451 R,452 P, 453 L, 455 F, 457 F, 458 P, 460 D, 463 T, 468 I, 469 Q, 470 F,471 L, 473 G, 477 M, 479 S, 480 P, 482 L, 485 G, 489 V, 491 A, 492 Y,494 P, 496 G, 497 N, 498 W, 501 L, 504 Y, 508 V, 513 G, 518 L, 521 P,523 D, 524 H, 526 N, 527 V, 528 H, 531 E, 532 G, 534 I, 537 M, 538 Q,539 G, 541 A, 543 T, 544 T, 547 A, 550 T, 554 L, 555 L, 556 V, 557 V,559 S, 566 G, 567 E, 568 L, 569 F, 571 D, 579 G, 583 G, 585 W, 586 T,588 V, 590 F, 603 S, 605 V, 606 V, 611 A, 620 K, 622 T, 625 G, 635 Y,658 F, 664 S, 669 L, 671 G, 674 F

and 678 L of SEQ ID NO: 2 and which comprises at least approximately1-69, preferably at least 139, in particular at least 194, morepreferably at least 249 and very especially preferably approximately263-274 amino acid residues which are selected from the group of theamino acid residues consisting of

1 P, 2 K, 3 L, 4 R, 5 P, 6 S, 7 V, 8 H, 9 P, 10 S, 11 Q, 12 H, 13 H, 14P, 15 I, 16 Q, 17 L, 19 R, 20 P, 21 P, 22 A, 23 L, 24 H, 27 Y, 28 S, 29F, 30 R, 31 Y, 32 F, 35 V, 36 S, 43 S, 49 I, 50 V, 57 S, 64 L, 84 Q, 91A, 109 I, 110 D, 112 V, 114 L, 118 S, 122 S, 152 E, 153 R, 154 V, 155 I,156 F, 158 L, 159 R, 163 Q, 165 D, 172 V, 178 I, 180 N, 183 D, 186 R,198 D, 199 N, 200 M, 203 Q, 205 V, 210 N, 221 T, 222 E, 223 V, 226 R,230 E, 231 K, 236 V, 238 F, 243 L, 259 S, 262 F, 275 H, 280 Y, 281 R,288 T, 293 T, 294 M, 311 Y, 312 S, 316 N, 317 V, 326 V, 331 L, 335 R,338 S, 360 S, 378 S, 404 K, 408 P, 413 S, 420 A, 422 K, 430 Q, 437 M,444 I, 445 K, 447 T, 461 A, 464 F, 465 D, 468 T, 478 I, 481 I, 487 T,510 L, 511 N, 512 Q, 516 M, 536 V, 548 Q, 549 R, 551 A, 553 K, 558 L,560 S, 561 S, 562 K, 570 V, 573 D, 574 D, 577 Q, 580 R, 581 E, 584 R,591 N, 592 S, 593 N, 594 I, 595 I, 598 K, 599 I, 601 V, 602 K, 609 R,612 L, 613 D, 615 G, 616 L, 618 L, 619 E, 623 L, 630 R, 631 G, 632 L,634 S, 637 L, 638 V, 639 G, 641 H, 642 Q, 643 Q, 644 G, 645 N, 646 T,647 T, 648 M, 649 K, 650 E, 651 S, 652 L, 653 K, 654 Q, 656 G, 657 Q,659 V, 660 T, 661 M, 665 M, 668 I, 670 I, 679 Y, 680 I, 681 I, 682 T,693 H, 700 R, 703 G, 705 H, 706 G, 707 V, 709 L, 710 L, 712 S, 713 N,714 G, 715 M, 716 D, 718 Y, 720 G, 721 R, 722 I, 724 Y, 726 V, 727 I,728 G, 729 G, 730 I, 731 D, 732 L, 733 Y, 734 F, 735 A, 736 G, 739 P,742 V, 743 Q, 745 T, 747 I, 748 G, 749 R, 750 P, 751 A, 753 M, 754 P,755 Y, 756 W, 757 F, 758 G, 759 F, 761 Q, 762 C, 763 R, 764 G, 765 Y,768 V, 769 V, 771 Y, 772 A, 775 I, 776 P, 777 L, 778 E, 779 V, 780 M,781 W, 782 T, 783 D, 784 I, 785 D, 786 Y, 787 M, 788 D, 789 K, 790 D,791 F, 792 T, 793 L, 794 D, 795 P, 796 V, 798 F, 799 P, 804 F, 806 L,807 H, 808 N, 810 Q, 812 Y, 813 V, 814 I, 816 D, 817 P, 818 G, 819 I,821 Y, 822 T, 824 R, 825 G, 826 M, 828 V, 829 F, 831 K

and 832 R (here identified by the single-letter code for amino acids) ofSEQ ID NO: 2.

The term “part” with regard to the α-glucosidase protein (polypeptide,amino acid sequence) according to the invention encompasses, for thepurposes of the present invention, a poly- or oligopeptide composed ofat least approximately 10-50, preferably at least 100, more preferablyat least 200, especially preferably at least 400 and most preferablyapproximately 550-675 of the amino acid residues of the α-glucosidaseencoded by the nucleic acid molecule according to the invention or itsderivatives.

The present invention furthermore relates to a nucleic acid moleculewhich comprises a nucleic acid molecule of SEQ ID NO: 1 in accordancewith the cDNA insert of the plasmid DSM No. 12347 deposited at the DSZMon Jul., 24, 1998, or its derivatives or parts, in particular of thecoding region or its derivatives or parts.

The term “derivative” with regard to the nucleic acid molecule(nucleotide sequence or polynucleotide) according to the inventionencompasses, for the purposes of this invention, a polynucleotide whichcomprises at least 478 nucleotides, preferably at least 668, inparticular at least 860, and very especially preferably approximately907-945 nucleotides selected from the group consisting of

4 A, 6A, 12 A, 13 C, 17 G, 25 C, 32 A, 34 C, 38 A, 45 T, 47 A, 49 C, 51T, 56 G, 62 C, 65 C, 68 T, 73 C, 76 G, 77 G, 78 A, 79 T, 97 G, 100 G,101 G, 106 A, 108 T, 109 C, 110 A, 111 T, 112G, 113 G, 114 G, 115 G, 116T, 119 T, 122 T, 125 T, 127 A, 130 A, 131 G, 132 C, 133 A, 134 A, 135 T,136 G, 137 G, 139 A, 140 T, 141 G, 142 G, 143 A, 144 T, 146 T, 151 T,152 A, 153 T, 157 G, 158 G, 161 A, 162 T, 164 G, 166 A, 167 T, 169 A,171 T, 172T, 173 A, 174 C, 175 A, 176 A, 178 G, 179 T, 181 A, 182 T, 183T, 184 G, 185 G, 187 G, 188 G, 191 T, 193 A, 194 T, 195 T, 196 G, 197 A,200 T, 202 T, 203 A, 206 T, 208 T, 209 T, 211 G, 212 C, 214 G, 215 G,216 A, 217 C, 221 C, 223 C, 224 C, 226 G, 232 G, 233 T, 236 T, 237 G,239 A, 241 C, 242 A, 243 G, 244 T, 247 A, 248 C, 249 T, 254 T, 256 A,257 T, 259 G, 260 G, 263 G, 265 C, 266 C, 268 G, 269 C, 272 C, 274 A,275 T, 276 G, 277 C, 278 C, 280 T, 281 A, 283 T, 284 G, 285 G, 289 T,290 T, 292 G, 293 G, 297 T, 298 C, 299 A, 301 C, 302 A, 304 T, 305 G,308 G, 310 T, 313 G, 314 G, 316 T, 317 A, 323 A, 324 T, 326 T, 331 G,332 A, 335 T, 338 A, 343 G, 344 T, 346 G, 347 T, 349 G, 355 T, 356 A,357 T, 358 G, 359 C, 360 A, 362 A, 365 C, 366 T, 370 A, 371 T, 373 C,374 C, 377 T, 379 G, 380 A, 382 G, 383 T, 385 A, 386 T, 387 G, 388 T,389 G, 390 G, 391 A, 392 C, 394 G, 395 A, 397 A, 398 T, 399 T, 400 G,401 A, 402 T, 403 T, 404 A, 406 A, 407 T, 408 G, 409 G, 410 A, 411 T,412 G, 415 T, 418 A, 419 A, 421 G, 422 A, 424 T, 425 T, 426 C, 427 A,428 C, 431 T, 433 G, 434 A, 436 C, 437 C, 439 G, 440 T, 443 A, 445 T,446 T, 448 C, 449 C, 454 G, 455 A, 459 G, 461 T, 469 T, 470 T, 471 T,473 T, 478 A, 481 C, 482 T, 484 C, 485 A, 489 G, 490 A, 491 A, 492 T,493 G, 496 C, 497 A, 499 A, 501 A, 502 T, 503 A, 505 G, 506 T, 511 A,512 T, 515 T, 517 G, 518 A, 519 T, 520 C, 521 C, 523 G, 524 G, 526 A,527 T, 536 A, 538 A, 542 C, 544 T, 545 A, 546 T, 547 G, 550 A, 551 C,553 T, 556 A, 559 A, 560 G, 562 G, 563 G, 565 A, 566 T, 567 G, 569 A,570 A, 575 A, 576 T, 577 G, 578 T, 580 T, 581 T, 584 T, 586 A, 587 A,590 G, 593 A, 594 T, 597 T, 601 C, 602 C, 604 T, 605 A, 607 C, 610 G,611 G, 616 G, 617 T, 619 T, 620 G, 621 G, 622 C, 623 C, 625 G, 626 G,631 G, 632 T, 634 T, 635 A, 637 T, 640 C, 641 C, 643 G, 644 A, 646 T,647 T, 650 T, 653 A, 655 C, 656 C, 659 C, 660 T, 662 C, 663 T, 670 T,671 T, 673 T, 674 G, 675 G, 680 A, 682 G, 683 A, 685 A, 686 T, 689 A,690 G, 694 T, 695 T, 697 C, 701 A, 704 T, 707 T, 709 C, 710 C, 713 T,715 G, 716 A, 717 T, 718 G, 719 G, 722 T, 724 T, 725 G, 726 G, 728 T,730 G, 731 A, 733 A, 734 T, 735 G, 736 A, 737 A, 739 G, 740 A, 745 T,746 C, 748 A, 749 A, 751 T, 752 T, 754 A, 755 T, 758 C, 759 T, 760 T,761 C, 764 C, 766 C, 773 C, 778 T, 779 C, 780 T, 782 C, 785 T, 787 G,788 A, 791 A, 792 T, 793 C, 794 C, 796 C, 797 C, 799 T, 800 A, 802 A,803 A, 805 A, 806 T, 808 A, 809 A, 811 A, 812 A, 814 T, 815 C, 817 G,818 G, 820 G, 829 C, 830 C, 832 A, 833 T, 835 A, 841 A, 844 A, 845 C,848 I, 850 C, 851 C, 854 C, 856 A, 857 C, 860 C, 862 A, 865 C, 866 A,868 T, 870 T, 871 G, 872 G, 875 A, 883 G, 884 A, 886 T, 887 A, 890 A,891 T, 892 G, 895 C, 896 A, 897 T, 898 A, 899 A, 902 T, 904 T, 906 T,907 G, 908 G, 914 T, 916 G, 917 A, 918 A, 920 C, 921 T, 924 A, 925 G,927 C, 928 A, 929 C, 937 G, 938 C, 941 T, 944 T, 953 C, 958 A, 962 G,964 C, 965 C, 967 T, 968 T, 971 T, 974 T, 979 A, 980 G, 982 T, 983 C,985 A, 986 C, 988 T, 989 T, 990 T, 995 G, 997 T, 998 C, 1000 G, 1001 G,1003 A, 1006 T, 1007 A, 1008 C, 1009 A, 1010 C, 1013 C, 1015 C, 1016 A,1018 T, 1019 G, 1020 G, 1021 A, 1022 C, 1024 G, 1025 G, 1027 G, 1028 A,1029 T, 1030 A, 1031 A, 1032 T, 1033 G, 1034 C, 1035 T, 1036 G, 1037 C,1039 A, 1042 T, 1043 G, 1044 G, 1046 A, 1048 G, 1049 A, 1052 T, 1057 T,1058 A, 1059 C, 1060 T, 1061 C, 1063 A, 1064 T, 1066 C, 1067 C, 1072 A,1073 T, 1076 T, 1080 C, 1081 T, 1082 T, 1083 T, 1084 G, 1085 G, 1088 T,1090 T, 1091 T, 1092 T, 1093 G, 1094 G, 1096 A, 1097 T, 1099 C, 1100 C,1102 A, 1103 T, 1104 G, 1106 T, 1108 G, 1109 G, 1111 G, 1112 C, 1114 G,1115 A, 1116 T, 1117 A, 1118 T, 1120 T, 1121 G, 1123 G, 1124 G, 1125 T,1126 T, 1127 T, 1138 A, 1139 C, 1141 A, 1142 C, 1144 G, 1145 A, 1147 G,1148 A, 1151 T, 1153 T, 1154 G, 1157 G, 1159 C, 1160 G, 1162 T, 1163 G,1164 G, 1165 A, 1166 T, 1168 C, 1169 A, 1170 G, 1171 C, 1172 T, 1174 G,1175 G, 1177 G, 1178 C, 1180 T, 1181 T, 1183 T, 1184 A, 1186 C, 1187 C,1189 T, 1190 T, 1193 C, 1195 A, 1196 G, 1198 G, 1199 A, 1201 C, 1202 A,1204 T, 1205 C, 1208 C, 1213 G, 1216 A, 1217 C, 1220 C, 1225 C, 1226 A,1228 G, 1229 A, 1231 C, 1232 T, 1234 T, 1235 A, 1240 T, 1241 G, 1242 G,1243 G, 1244 A, 1246 T, 1247 C, 1249 G, 1250 T, 1252 G, 1253 C, 1254 T,1256 C, 1259 C, 1261 G, 1262 C, 1264 A, 1267 A, 1270 G, 1271 T, 1274 T,1276 G, 1277 G, 1279 C, 1280 T, 1281 C, 1283 G, 1292 T, 1294 C, 1295 T,1297 C, 1298 C, 1301 A, 1306 T, 1307 A, 1309 A, 1313 T, 1315 A, 1316 T,1317 G, 1318 T, 1319 A, 1321 G, 1322 A, 1324 G, 1325 C, 1328 A, 1331 T,1333 A, 1336 G, 1337 G, 1339 A, 1342 C, 1343 C, 1345 A, 1346 T, 1348 G,1349 C, 1351 C, 1352 G, 1354 C, 1355 C, 1357 C, 1358 T, 1360 T, 1362 C,1363 T, 1364 T, 1367 C, 1369 T, 1370 T, 1372 C, 1373 C, 1376 A, 1378 G,1379 A, 1387 A, 1388 C, 1390 T, 1393 G, 1396 A, 1397 T, 1403 C, 1405 C,1406 A, 1407 G, 1408 T, 1409 T, 1412 T, 1415 T, 1417 G, 1418 G, 1420 A,1422 A, 1424 G, 1427 T, 1429 A, 1430 T, 1431 G, 1433 T, 1435 T, 1436 C,1438 C, 1439 C, 1444 C, 1445 T, 1448 A, 1450 C, 1453 G, 1454 G, 1456 G,1463 C, 1465 G, 1466 T, 1470 T, 1471 G, 1472 C, 1474 T, 1475 A, 1477 T,1480 C, 1481 C, 1486 G, 1487 G, 1488 A, 1489 A, 1490 A, 1492 T, 1493 G,1494 G, 1496 T, 1501 C, 1502 T, 1504 T, 1508 A, 1510 T, 1511 A, 1514 C,1520 C, 1522 G, 1523 T, 1527 T, 1533 T, 1537 G, 1538 G, 1540 A, 1544 A,1547 T, 1549 A, 1552 C, 1559 C, 1561 C, 1562 C, 1565 C, 1567 G, 1568 A,1569 T, 1570 C, 1571 A, 1575 T, 1577 A, 1578 A, 1579 G, 1580 T, 1582 C,1583 A, 1586 T, 1588 C, 1591 G, 1592 A, 1593 A, 1594 G, 1595 G, 1597 A,1600 A, 1601 T, 1604 T, 1605 G, 1606 G, 1609 A, 1610 T, 1611 G, 1612 C,1613 A, 1614 A, 1615 G, 1616 G, 1619 A, 1621 G, 1622 C, 1625 T, 1626 G,1627 A, 1628 C, 1630 A, 1631 C, 1636 G, 1639 G, 1640 C, 1645 A, 1648 A,1649 C, 1652 C, 1654 T, 1658 A, 1660 C, 1661 T, 1664 T, 1665 G, 1667 T,1669 G, 1670 T, 1675 A, 1676 G, 1689 C, 1690 A, 1692 C, 1696 G, 1697 G,1699 G, 1700 A, 1703 T, 1705 T, 1706 T, 1709 T, 1711 G, 1712 A, 1715 A,1717 G, 1724 A, 1727 T, 1732 A, 1733 T, 1735 G, 1736 G, 1745 G, 1746 A,1747 G, 1748 G, 1750 A, 1753 T, 1754 G, 1755 G, 1756 A, 1757 C, 1760 T,1762 G, 1763 T, 1765 A, 1768 T, 1769 T, 1775 G, 1776 C, 1781 T, 1783 A,1789 A, 1796 T, 1802 T, 1807 T, 1808 C, 1809 A, 1810 G, 1811 A, 1813 G,1814 T, 1816 G, 1817 T, 1828 T, 1830 T, 1831 G, 1832 C, 1836 G, 1845 A,1846 T, 1848 G, 1851 C, 1853 T, 1855 G, 1858 A, 1859 A, 1862 T, 1864 A,1865 C, 1868 T, 1871 T, 1873 G, 1874 G, 1876 T, 1877 T, 1881 A, 1890 A,1894 T, 1897 A, 1901 G, 1908 G, 1925 A, 1929 A, 1945 A, 1953 T, 1958 A,1966 G, 1972 T, 1973 T, 1976 T, 1984 G, 1988 T, 1990 T, 1991 C, 1997 T,2006 T, 2009 T, 2011 G, 2012 G, 2020 T, 2021 T, 2024 A, 2027 T

and 2033 T of SEQ ID NO: 1 and which furthermore comprises at leastapproximately 1-93 nucleotides, preferably at least 187, in particular261, more preferably at least 336 and very especially preferablyapproximately 354-369 nucleotides selected from the group consisting of

1 C, 10 A, 16 A, 19 G, 21 T, 23 A, 24 C, 26 C, 30 A, 33 A, 36 C, 39 T,43 A, 48 G, 52 C, 53 A, 54 C, 57 T, 58 C, 59 C, 60 G, 63 G, 64 G, 66 G,67 C, 69 C, 70 C, 71 A, 72 C, 74 G, 75 G, 80 A, 81 C, 86 T, 88 C, 89 G,91 T, 93 C, 94 T, 96 C, 99 C, 102 A, 103 G, 104 T, 105 T, 107 G, 123 T,128 G, 138 C, 145 A, 149 T, 156 G, 170 G, 189 G, 190 T, 192 A, 199 T,201 G, 264 T, 271 G, 279 A, 291 C, 294 A, 309 G, 325 A, 327 T, 328 G,333 T, 334 G, 340 C, 341 T, 342 G, 348 G, 354 T, 363 G, 364 T, 375 G,384 T, 420 G, 429 A, 432 C, 438 A, 444 C, 456 G, 457 C, 458 G, 460 G,462 A, 464 T, 465 T, 467 T, 468 T, 472 C, 476 G, 477 G, 480 G, 486 T,487 C, 494 A, 507 A, 513 A, 514 G, 516 A, 522 A, 525 A, 534 C, 537 C,540 T, 543 A, 549 C, 552 C, 557 G, 558 G, 564 C, 573 A, 592 G, 595 A,596 A, 599 T, 603 C, 608 A, 609 A, 612 G, 614 T, 627 G, 636 T, 639 T,651 A, 661 A, 665 A, 667 G, 668 T, 669 A, 678 A, 687 T, 688 G, 692 A,706 G, 708 A, 727 C, 744 G, 771 A, 774 A, 775 T, 776 C, 783 C, 784 T,798 C, 807 A, 813 C, 819 C, 825 C, 834 C, 838 T, 842 G, 843 A, 855 C,858 T, 863 C, 864 A, 878 C, 879 A, 881 T, 882 G, 885 G, 888 T, 903 T,912 A, 931 T, 934 A, 935 G, 940 T, 949 G, 954 T, 957 T, 976 G, 971 T,987 T, 991 C, 1002 C, 1004 G, 1012 T, 1038 T, 1041 C, 1062 C, 1068 T,1079 G, 1087 T, 1095 A, 1132 A, 1140 T, 1161 C, 1167 T, 1179 A, 1188 A,1203 C, 1206 T, 1210 A, 1211 A, 1212 G, 1215 C, 1223 C, 1224 C, 1239 T,1258 G, 1263 C, 1265 A, 1275 T, 1278 G, 1287 T, 1288 C, 1291 T, 1293 A,1296 T, 1305 T, 1310 T, 1311 G, 1312 C, 1314 T, 1330 A, 1338 G, 1340 C,1341 T, 1344 C, 1347 T, 1353 A, 1356 C, 1386 G, 1389 A, 1391 T, 1402 A,1416 C, 1432 A, 1441 A, 1443 A, 1446 T, 1455 A, 1459 A, 1460 C, 1461 C,1467 T, 1497 T, 1500 C, 1503 C, 1518 C, 1521 T, 1528 T, 1530 G, 1531 A,1534 C, 1535 A, 1546 A, 1557 C, 1563 A, 1566 A, 1575 A, 1581 A, 1590 T,1596 G, 1602 A, 1603 T, 1607 T, 1608 C, 1632 A, 1641 T, 1643 A, 1644 G,1650 T, 1651 G, 1653 A, 1657 A, 1659 A, 1665 T, 1668 C, 1672 C, 1678 A,1680 C, 1681 A, 1683 C, 1684 A, 1695 A, 1698 A, 1704 A, 1708 G, 1718 A,1719 C, 1738 A, 1743 G, 1749 G, 1751 G, 1752 G, 1758 G, 1761 A, 1772 A,1773 C, 1774 A, 1784 T, 1786 T, 1788 C, 1791 T, 1792 A, 1795 A, 1800 G,1801 G, 1803 T, 1805 A, 1812 G, 1815 T, 1825 C, 1834 C, 1837 G, 1842 A,1843 G, 1847 T, 1852 C, 1857 A, 1869 A, 1875 A, 1878 T, 1884 T, 1886 T,1891 G, 1895 T, 1896 G, 1902 C, 1903 T, 1904 A, 1905 T, 1906 G, 1909 C,1911 T, 1913 T, 1914 T, 1915 G, 1918 T, 1919 C, 1920 A, 1922 A, 1923 C,1924 C, 1932 G, 1936 A, 1940 C, 1948 G, 1949 A, 1955 T, 1957 A, 1959 G,1960 C, 1962 G, 1964 G, 1969 C, 1975 G, 1979 C, 1981 A, 1986 A, 1989 C,1995 G, 1996 A, 2001 A, 2002 A, 2005 T, 2007 G, 2008 A, 2035 T, 2038 A,2040 C, 2042 T, 2044 A, 2045 C, 2046 T, 2047 T and 2048 A of SEQ ID NO:1.

In the numbering of the positions of the individual elements of thenucleotide or amino acid sequences according to the invention of SEQ IDNO: 1 or SEQ ID NO: 2, which has been stated above explicitly,derivatives of said sequences according to the invention are also to beunderstood as meaning those sequences in which the numbering of theindividual sequence elements may deviate from those of the SEQ ID NOS: 1or 2 according to the invention. What is decisive here is significantagreement of at least one sequence section (“part”) with the sequenceaccording to the invention. Such agreements can be determined in asimple manner using general expert knowledge, for example by making useof suitable computer programs, for example by carrying out a sequencecomparison of the sequence according to the invention with a sequence inquestion to be compared (so-called sequence alignment). Such computerprograms, which, for example, are commercially available (for exampleOmiga®, Version 1.1.3. by Oxford Molecular Ltd., Oxford, UK) and whichin some cases are also an integral component of sequence databases (forexample EMBL, GenBank), identify, for example, the best-possibleagreement of identical, or, if appropriate, chemical equivalent,sequence elements and take into consideration in particular theexistence of insertions and/or deletions which may lead to a shift ofindividual sequence elements or of sequence sections and which can thusaffect numbering of the sequence elements or sequence sections.

With regard to the nucleic acid molecule according to the inventionwhich encodes an α-glucosidase, the term “derivative” furthermoreencompasses a nucleic molecule which deviates from SEQ ID NO: 1 byaddition, deletion, insertion or recombination of one or morenucleotides and which meets the conditions as defined above.

With regard to the nucleic acid molecule according to the inventionwhich encodes an α-glucosidase, the term “derivative” furthermorecomprises a complementary or inverted-complementary sequence(polynucleotide) of the nucleic acid molecule according to the inventionor of derivatives or parts thereof.

The term “part”, which refers to the nucleic acid molecule according tothe present invention which encodes an α-glucosidase, encompasses apoly- or oligonucleotide composed of at least approximately 15-35,preferably at least approximately 36-100, in particular at least 200,more preferably at last 400, especially preferably at least 800 and mostpreferably approximately 1400-1700 of the nucleotides of a nucleic acidmolecule according to the invention which encodes an α-glucosidase, ortheir derivatives.

In a preferred embodiment of the present invention, the terms“derivative” and/or “part” according to the present invention encompassa polynucleotide, or a poly- or oligopeptide as defined above, whichshows functional and/or structural equivalence of the α-glucosidase geneobtained from potato (i.e. of the nucleic acid molecule which encodesthe α-glucosidase) or α-glucosidase polypeptide. The term “functionaland/or structural equivalence” generally means the same, an equivalentor similar function of the inventive molecule in question, ifappropriate especially biological function.

The invention furthermore relates to a recombinant nucleic acid moleculecomprising a) a nucleotide sequence encoding a protein with the functionof an α-glucosidase, preferably from potato, or parts of said nucleotidesequence, and b) one or more nucleotide sequences which encode a proteinselected from amongst group A, composed of proteins with the function ofbranching enzymes, ADP glucose pyrophosphorylases, granule-bound starchsynthases, soluble starch synthases, debranching enzymes,disproportioning enzymes, plastid starch phosphorylases, R1 enzymes,amylases, glucosidases, parts of nucleotide sequences encoding proteinsselected from amongst group A and nucleic acid molecules which hybridizewith one of said nucleotide sequences or parts thereof, preferably adeoxyribonucleic acid or ribonucleic acid molecule, especiallypreferably a cDNA molecule. Especially preferred is a nucleic acidmolecule which specifically hybridizes with one of said nucleotidesequences or parts thereof.

The nucleotide sequence according to the invention encoding a proteinwith the function of a potato α-glucosidase is depicted by SEQ ID NO: 1,the protein encoded by the nucleotide sequence by SEQ ID NO:2.

SEQ ID NO: 1cgaatacgaataaccgacgctaaccatcaacgatgggaagtgccggaagaaattctccaccgtccaccaccgccgtcgccgccgtcaacctccaactcctcatcagaaaaccactccccaattaccctctctaacccaaactcagacctagagttcacccttcacaacaccatcccattcagcttcaccgtccgccggcgctccaccggggatactcttttcgatacttcgccggagttagtcatggggttttgcttctgagtagcaatggcatggatattgtgtatacgggtgataggattagttacaaggtgattggagggttaattgatttgtatttctttgccggaccttcgccggaaatggtggtggatcagtatactcagcttattggtcgtcctgctgctatgccatattggtctttcggatttcaccaatgccggtggggttacaagaatattgatgatgttgaactggtagtggatagttatgcaaagtctagaataccgctggaggttatgtggactgatattgattacatggatggttttaaggacttcacactcgatccagttaacttcccactggagcgggtaattttttttctcaggaagcttcatcagaatgatcagaaatatgtactaatagtagatccaggaattagcatcaacaatacatatgacacctataggagaggcatggaagcagatgtcttcataaaacgcgataatatgccctaccaaggggttgtttggccagggaatgtttattatcctgattttctaaatccagctactgaagtattttggagaaatgaaattgagaagttccaggatctcgtaccttttgatggcctgtggcttgacatgaatgaattgtcaaacttcataacttcccctcctacaccatcatctacctttgatgatcctccctacaagataaacaactctggcgatcacttgcccatcaattatagaacagttccagccacttctacacattttggtgatacaatggagtataatgtccataacctttatggattacttgaatctagagccacttatagtgcattggttaatgtcactggtaaaaggccattcattcttgtaagatcaacttttcttggctctggcagatacacgtcacattggactggagataatgctgctacctggaacgatttggcatactccattcctactatcttgagctttggattgtttggaattccaatggttggagctgatatatgtggtttttcaagtaacactactgaagagctttgccgccgctggattcagcttggagcattctatccatttgcaagagaccactctgctaaggacacaaccccccaagagctctatagttgggattcagttgctgcagcagccaagaaagtccttgggctccgatatcagttacttccatacttttatatgcttatgtacgaggcacatataaaagggactcccattgcacgacccctcttcttctctttccctcaagatgccaagacatttgatatcagcacacagttccttctcggtaaaggtgtcatgatctcacctatacttaagcaaggagcaacctctgttgatgcatatttccctgctggaaactggtttgacctcttcaattactctcgctctgtgagtttgaatcaaggaacatatatgacacttgacgcaccaccagatcatataaatgtacatgttcgtgaagggaacatattggtcatgcaaggggaagcaatgacaacacaagctgctcagaggactgcattcaaactccttgtcgtgctgagcagcagcaaaaacagcacaggagaactatttgtggacgatgacgatgaggtgcagatgggaagagagggagggaggtggacgctagttaagtttaacagcaatatcattggcaataaaattgtggttaaatcagaggttgtgaatggacgatatgcgctggatcaaggattggtccttgaaaaggtgacattattgggatttgaaaatgtgagaggattgaagagctatgagcttgttggatcacaccagcaagggaacacaacaatgaaggaaagtcttaagcagagtggacagtttgttactatggaaatctcagggatgtcaatattgatagggaaagagttcaaattggagctatacatcattactaacaaatgaattaagttatatacgcttgttgtatgaaattttctttcatttatcaatgcagtttaatttatgataaaaaaaaaaaaaaaaa SEQ ID NO: 2PKLRPRVHPSQHHPIQLHRPPALHRGYSFRYFAGVSHGVLLLSSNGMDIVYTGDRISYKVIGGLIDLYFFAGPSPEMVVDQYTQLIGRPAAMPYWSFGFHQCRWGYKNIDDVELVVDSYAKSRIPLEVMWTDIDYMDGFKDFTLDPVNFPLERVIFFLRKLHQNDQKYVLIVDPGISINNTYDTYRRGMEADVFIKRDNMPVQGVVWPGNVYYPDFLNPATEVFWRNEIEKFQDLVPFDGLWLDMNELSNFITSPPTPSSTFDDPPYKINNSGDHLPINYRTVPATSTHFGDTMEYNVHNLYGLLESRATYSALVNVTGKRPFILVRSTFLGSGRYTSHWTGDNAATWNDLAYSIPTILSFGLFGIPMVGADICGFSSNTTEELCRRWIQLGAFYPFARDHSAKDTTPQELYSWDSVAAAAKKVLGLRYQLLPYFYMLMYEAHIKGTPIARPLFFSFPQDAKTFDISTQFLLGKGVMISPILKQGATSVDAYFPAGNWFDLFNYSRSVSLNQGTYMTLDAPPDHINVHVREGNILVMQGEAMTTQAAQRTAFKLLVVLSSSKNSTGELFVDDDDEVQMGREGGRWTLVKFNSNIIGNKIVVKSEVVNGRYALDQGLVLEKVTLLGFENVRGLKSYELVGSHQQGNTTMKESLKQSGQFVTMEISGMSILIGKEFKLELYIIT

The α-glucosidase nucleotide sequence according to the invention showsrelatively little sequence homology with known α-glucosidase-encodingmolecules (Taylor et al., 1998, Plant J. 13: 419-424, Sugimoto et al.,1997, Plant Mol. Biol. 33, 765-768; EMBL Datenbank-Einträge: U22450,P10253, D86624). The amino acid sequence differs markedly from theα-glucosidases described in the prior art, in particular in the 5′region, as can be seen from a sequence alignment with SEQ ID NO: 2.

Nucleotide sequences which encode a protein of group A and which aresuitable according to the invention have been described, for example,for soluble (types I, II, III or IV) or granule-bound starch synthaseisoforms in Hergersberg, 1988, Ph.D. thesis, University of Cologne;Abel, 1995, Ph.D. thesis, FU Berlin; Abel et al., 1996, Plant Journal10(6):981-991; Visser et al., 1989, Plant Sci. 64:185-192; van der Leijet al., 1991, Mol. Gen. Genet. 228:240-248; EP-A-0779363; WO 92/11376;WO 96/15248; WO 97/26362; WO 97/44472; WO 97/45545; Delrue et al., 1992,J. Bacteriol. 174: 3612-3620; Baba et al., 1993, Plant Physiol.103:565-573; Dry et al., 1992, The Plant Journal 2,2: 193-202 or else inthe EMBL database entries X74160; X58453; X88789; X 94400; for branchingenzyme isoforms (branching enzymes I, IIa or IIb), debranching enzymeisoforms (debranching enzyme, isoamylases, pullulanases, R1 enzymes) ordisproportioning enzyme isoforms, for example, described in WO 92114827;WO 95107335; WO 95/09922; WO 96/19581; WO 97/22703; WO 97/32985; WO97/42328; Takaha et al., 1993, J. Biol. Chem. 268: 1391-1396 or else inthe EMBL database entry X83969, and those for ADP glucosepyrophosphorylases and plastid starch phosphorylase isoforms, forexample, described in EP-A-0368506; EP-A-0455316; WO 94/28146; DE19653176.4; WO 97/11188; Brisson et al., 1989, The Plant Cell 1:559-566;Buchner et al., 1996, Planta 199:64-73; Camirand et al., 1989, PlantPhysiol. 89(4 Suppl.) 61; Bhatt & Knowler, J. Exp. Botany 41 (Suppl.)5-7; Lin et al., 1991, Plant Physiol. 95: 1250-1253; Sonnewald et al.,1995, Plant Mol. Biol. 27:567-576; DDBJ No. D23280; Lorberth et al.,1998, Nature Biotechnology 16:473-477.

The nucleotide sequences to be employed suitably in accordance with theinvention are of pro- or eukaryotic origin, preferably of bacterial,fungal or plant origin.

The term “parts of nucleotide sequences” denotes, for the purposes ofthe present invention, parts of the nucleotide sequences to be used inaccordance with the invention which are at least 15 bp, preferably atleast 150 bp, especially preferably at least 500 bp in length, but whichdo not exceed a length of 5000 bp, preferably 2500 bp.

The term “hybridization” means, for the purposes of the presentinvention, hybridization under conventional hybridization conditions,preferably under stringent conditions, as are described, for example, inSambrook et al., Molecular Cloning, A Laboratory Manual, 2nd Ed. (1989)Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y.).

A “specific hybridization” especially preferably takes place under thefollowing highly stringent conditions: Hybridization buffer: 2×SSC;10×Denhardt solution (Fikoll 400+PEG+BSA; ratio 1:1:1); 0.1% SDS; 5 mMEDTA; 50 mM Na₂HPO₄; 250 μg/ml herring sperm DNA; 50 μg/ml tRNA; or 0.25M sodium phosphate buffer pH 7.2; 1 mM EDTA; 7% SDS at a

Hybridization temperature: T = 55 to 68° C., Wash buffer: 0.2 × SSC;0.1% SDS and Wash temperature: T = 40 to 68° C..

The molecules which hybridize with the nucleic acid molecules accordingto the invention also encompass fragments, derivatives and allelicvariants of the nucleic acid molecules according to the invention.Fragments are to be understood as meaning parts of the nucleic acidmolecules which are long enough to encode a functionally active part ofthe proteins described. The term derivative means in this context thatthe sequences of these molecules differ from the sequences of thenucleic acid molecules according to the invention in one or morepositions and exhibit a high degree of homology to these sequences.Homology means a sequence identity of at least 60%, preferably over 70%and especially preferably over 85%. The deviations relative to thenucleic acid molecules according to the invention may have originated bymeans of deletions, substitutions, insertions or recombinations.

Homology furthermore means that functional and/or structural equivalenceexists between the nucleic acid molecules in question or the proteinsencoded by them. The nucleic acid molecules which are homologous to themolecules according to the invention and which constitute derivatives ofthese molecules are, as a rule, variations of these molecules whichconstitute modifications which exert the same biological function. Theymay be naturally occurring variations, for example sequences from otherplant species, or mutations, it being possible for these mutations tohave occurred naturally or to have been introduced by directedmutagenesis. The variations may further be synthetic sequences. Theallelic variants may be naturally occurring variants or else syntheticvariants or variants generated by recombinant DNA technology.

The nucleic acid molecules according to the invention may be DNAmolecules, in particular cDNA or genomic molecules. The nucleic acidmolecules according to the invention may furthermore be RNA molecules.The nucleic acid molecules according to the invention or parts thereofcan have been obtained, for example, from natural sources or generatedby means of recombinant technology or by synthesis.

To express the nucleic acid molecules according to the invention insense or antisense orientation in plant cells, they are linked toregulatory DNA elements which ensure transcription in plant cells. Theseinclude, in particular, promoters. In general, any promoter which isactive in plant cells is suitable for expression. The promoter may havebeen chosen in such a way that expression is constitutive or only in aparticular tissue, at a particular point in time of plant development orat a point in time determined by external factors which can be, forexample, chemically or biologically inducible. Relative to thetransformed plant, the promoter—and also the nucleotide sequence—can behomologous or heterologous. Examples of suitable promoters are thecauliflower mosaic virus 35S RNA promoter for constitutive expression,the patatin gene promoter B33 (Rocha-Sosa et al., 1989, EMBO J. 8:23-29)for tuber-specific expression in potatoes or a promoter which ensuresexpression only in photosynthetically active tissues, for example theST-LS1 promoter (Stockhaus et al., 1987, Proc. Natl. Acad. Sci. USA 84:7943-7947; Stockhaus et al., 1989, EMBO J. 8: 2445-2451) or, forendosperm-specific expression, the wheat HMG promoter or promoters frommaize zein genes.

A termination sequence which terminates the nucleic acid moleculeaccording to the invention may serve to correctly end transcription andto add to the transcript a poly-A tail, which is considered to have afunction in stabilizing the transcripts. Such elements have beendescribed in the literature (cf. Gielen et al., 1989, EMBO J. 8:23-29)and are exchangeable as desired.

The nucleic acid molecules according to the invention can be used forgenerating transgenic plant cells and plants which show an increase orreduction in the activity of α-glucosidase or in the activity ofα-glucosidase and at least one further enzyme of starch metabolism. Tothis end, the nucleic acid molecules according to the invention areintroduced into suitable vectors, provided with the regulatory nucleicacid sequences which are necessary for efficient transcription in plantcells, and introduced into plant cells. On the one hand, there is thepossibility of using the nucleic acid molecules according to theinvention for inhibiting the synthesis of the endogenous α-glucosidaseor the endogenous α-glucosidase and at least one further protein ofgroup A in the cells. This can be achieved with the aid of antisenseconstructs, in-vivo mutagenesis, a cosuppression effect which occurs, orwith the aid of suitably constructed ribozymes. On the other hand, thenucleic acid molecules according to the invention can be used forexpressing α-glucosidase or α-glucosidase and at least one furtherprotein of group A in the cells of transgenic plants and thus lead to anincreased activity in the cells of the enzymes which have been expressedin each case.

In addition, there exists the possibility of using the nucleic acidmolecules according to the invention for inhibiting the synthesis of theendogenous α-glucosidase and the overexpression of at least one furtherprotein of group A in the cells.

Finally, the nucleic acid molecules according to the invention can alsobe used for expressing α-glucosidase and inhibiting at least one furtherprotein of group A in the cells of transgenic plants. The twolast-mentioned embodiments of the invention thus lead, in the cells, toa simultaneous inhibition and increase in the activities of the enzymeswhich are inhibited or expressed, respectively.

The invention furthermore relates to a vector comprising a nucleic acidmolecule according to the invention.

The term “vector” encompasses plasmids, cosmids, viruses, bacteriophagesand other vectors conventionally used in genetic engineering whichcontain the nucleic acid molecules according to the invention and whichare suitable for transforming cells. Such vectors are preferablysuitable for transforming plant cells. Especially preferably, theypermit integration of the nucleic acid molecules according to theinvention, if appropriate together with flanking regulatory regions,into the genome of the plant cell. Examples are binary vectors, such aspBinAR or pBinB33, which can be employed in agrobacteria-mediated genetransfer.

In a preferred embodiment, the vector according to the invention isdistinguished by the fact that the nucleotide sequence encoding aprotein with the function of an α-glucosidase or parts thereof ispresent in sense or antisense orientation.

In a further preferred embodiment, the vector according to the inventionis distinguished by the fact that the nucleotide sequence which encodesone or more proteins selected from amongst group A or parts thereof ispresent in sense or antisense orientation.

In yet a further preferred embodiment, the vector according to theinvention is distinguished by the fact that the nucleotide sequencewhich encodes a plurality of proteins selected from group A or partsthereof is present partly in sense and partly in antisense orientation.

Very especially preferably, the vector according to the invention islinked to regulatory elements which ensure expression in a prokaryoticor eukaryotic cell, i.e., for example, transcription and synthesis of anRNA which, if the nucleotide sequence is present in sense orientation,is translatable.

In addition, it is possible to introduce, by means of customarytechniques of molecular biology (see, for example, Sambrook et al.,1989, Molecular Cloning, A Laboratory Manual, 2nd Ed. Cold Spring HarborLaboratory Press, Cold Spring Harbour, N.Y.), various mutations into theDNA sequences according to the invention, which leads to the synthesisof proteins with biological properties which may have been modified. Onthe one hand, it is possible to generate deletion mutants in whichsequences are generated, by progressive deletions from the 5′ or the 3′end of the coding DNA sequences which lead to the synthesis ofanalogously truncated proteins. For example, such deletions at the 5′end of the DNA sequence allow the targeted production of enzymes which,due to the removal of the relevant transit or signal sequences, are nolonger localized in their original (homologous) compartment, but in thecytosol, or which, due to the addition of other signal sequences, arelocalized in one or more other (heterologous) compartments.

On the other hand, it is also feasible to introduce point mutations inpositions where an altered amino acid sequence affects, for example, theenzyme activity or the regulation of the enzyme. Thus, it is possible,for example, to generate mutants which have an altered K_(M) or k_(cat)value or which are no longer subject to the regulatory mechanismsnormally present in the cell via allosteric regulation or covalentmodification.

For the purposes of recombination manipulation in prokaryotic cells, theDNA sequences according to the invention or parts of these sequences canbe introduced into plasmids which permit mutagenesis or an alteredsequence by the recombination of DNA sequences. Base exchanges may beperformed or natural or synthetic sequences may be added, with the aidof standard methods in molecular biology (cf. Sambrook et al., 1989,loc. cit.). To link the DNA fragments to each other, adapters or linkersmay be attached to the fragments. Furthermore, manipulations whichprovide suitable restriction cleavage sites or which remove excessiveDNA or restriction cleavage sites which are no longer needed may beemployed. Where insertions, deletions or substitutions are suitable,in-vitro mutagenesis, primer repair, restriction or ligation may beused. The analytical methods which are generally employed are sequenceanalysis, restriction analysis and, if appropriate, other methods ofbiochemistry and molecular biology.

The invention furthermore relates to a host cell, in particularprokaryotic or eukaryotic cells, preferably bacterial or plant cells(for example from E. coli, Agrobacterium, Solananceae, Poideae, rye,barley, oats, maize, wheat, sorghum and millet, sago, rice, peas,marrowfat peas, cassava, potatoes, tomatoes, oilseed rape, soybeans,hemp, flax, sunflowers, cowpeas, mung beans, beans, bananas orarrowroot) which contains a nucleic acid molecule according to theinvention or a vector according to the invention or which is derivedfrom a cell which has been transformed with a nucleic acid moleculeaccording to the invention or a vector according to the invention.

The invention furthermore relates to host cells, in particularprokaryotic or eukaryotic cells, preferably bacterial or plant cells(for example of E. coli, Agrobacterium, Solanaceae, Poideae, rye,barley, oats, maize, wheat, sorghum and millet, sago, rice, peas,marrowfat peas, cassava, potatoes, tomatoes, oilseed rape, soybeans,hemp, flax, sunflowers, cowpeas, mung beans, beans, bananas orarrowroot) which contains, in addition to a recombinant nucleic acidmolecule encoding a protein with the function of a β-amylase, one ormore further recombinant nucleic acid molecules which encode a proteinselected from group A or their parts or nucleotide sequences hybridizingwith these nucleic acid molecules.

In addition to using the nucleic acid molecules according to theinvention, the host cells according to the invention may, ifappropriate, also be generated by successive transformation (so-calledsupertransformation), by employing individual nucleotide sequences orvectors comprising nucleotide sequences which encode a protein with thefunction of branching enzymes, ADP glucose pyrophosphorylases,granule-bound starch synthases, soluble starch synthases I, II, III orIV, debranching enzymes, disproportioning enzymes, plastid starchphosphorylases, R1 enzymes, amylases, glucosidases, parts thereof, andnucleic acid molecules which hybridizes with one of said nucleotidesequences or their parts, in a plurality of successive celltransformations. A further embodiment of the present invention relatesto a method of generating a transgenic plant cell which synthesizes amodified starch, which comprises integrating a nucleic acid moleculeaccording to the invention or a vector according to the invention intothe genome of a plant cell.

Providing the nucleic acid molecules according to the invention makes itpossible to engage in the start metabolism of plants, with the aid ofrecombinant methods, and to alter it in such a way that the result isthe synthesis of a modified starch which is altered relative to thestarch synthesized in the wild-type plant with regard to, for example,structure, water content, protein content, lipid content, fiber content,ash/phosphate content, amylase/amylopectin ratio, molecular massdistribution, degree of branching, granule size, granule shape andcrystallization, or else in its physico-chemical properties such as theviscoelasticity, the sorptive characteristics, gelatinizationtemperature, viscosity, thickening capacity, solubility, gel structure,transparency, thermal stability, shear stability, stability to acids,tendency to undergo retrogradation, gelling, freeze-thaw stability,complex formation, iodine binding, film formation, adhesion power,enzyme stability, digestibility or reactivity. There is also thepossibility of increasing the yield in suitably genetically modifiedplants by increasing the activity of proteins which are involved instarch metabolism, for example by overexpressing suitable nucleic acidmolecules, or by providing mutants which are no longer subject to thecell's regulatory mechanisms and/or which exhibit different temperaturedependencies relating to their activity. A particularly pronouncedincrease in yield may be the result of increasing the activity of one ormore proteins which are involved in the starch metabolism in specificcells of the starch-storing tissue of transformed plants such as, forexample, in the tuber in the case of potatoes or in the endosperm ofmaize or wheat. The economic importance and the advantages of thesepossibilities of engaging in the starch metabolism are obvious.

When expressing the nucleic acid molecules according to the invention inplants it is possible, in principle, for the protein synthesized to belocalized in any desired compartment of the plant cell. To achievelocalization in a particular compartment (cytosol, vacuole, apoplast,plastids, mitochondria), the transit or signal sequence which ensureslocalization must, if necessary, be deleted (removed) and the remainingcoding region must, if necessary, be linked to DNA sequences whichensure localization in the compartment in question. Such sequences areknown (see, for example, Braun et al., EMBO J. 11 (1992), 3219-3227;Wolter et al., Proc. Natl. Acad. Sci. USA 85 (1988), 846-850; Sonnewaldet al., Plant J. 1 (1991), 95-106).

The generation of plant cells with a reduced activity of a proteininvolved in the starch metabolism can be achieved, for example, byexpressing a suitable antisense RNA, a sense RNA for achieving acosuppression effect, in-vivo mutagenesis or by expressing a suitablyconstructed ribozyme which specifically cleaves transcripts which encodeone of the proteins involved in starch metabolism, using a nucleic acidmolecule according to the invention, preferably by expressing anantisense transcript.

To this end, it is possible to use, firstly, a DNA molecule whichencompasses all of the sequence which encodes a protein involved instarch metabolism including any flanking sequences, as well as DNAmolecules which only encompass parts of the coding sequence, these partshaving a minimum length of 15 bp, preferably of at least 100-500 bp, andin particular over 500 bp. As a rule, DNA molecules are used which areshorter than 5000 bp, preferably shorter than 2500 bp.

It is also possible to use DNA sequences which exhibit a high degree ofhomology to the sequences of the DNA molecules according to theinvention, but are not fully identical with them. The minimum homologyshould exceed approx. 65%. The use of sequences with a homology of 75%and in particular 80% is to be preferred.

The expression of ribozymes for reducing the activity of specificproteins in cells is known to the skilled worker and described, forexample, in EP-B10 321 201. The expression of ribozymes in plant cellswas described, for example, in Feyter et al. (Mol. Gen. Genet. 250(1996), 329-338).

Furthermore, the reduction of the proteins involved in the starchmetabolism in the plant cells according to the invention can also beachieved by so-called “in-vivo mutagenesis”, where an RNA-DNA hybridoligonucleotide (“chimeroplast”) is introduced into cells by celltransformation (Kipp P. B. et al., Poster Session at the “5thInternational Congress of Plant Molecular Biology, 21-27, Sept. 1997,Singapore; R. A. Dixon and C. J. Amtzen, Meeting report on “MetabolicEngineering in Transgenic Plants”, Keystone Symposia, Copper Mountain,Colo., USA, TIBTECH 15 (1997), 441-447; international patent applicationWO 95/15972; Kren et al., Hepatology 25 (1997), 1462-1468; Cole-Strausset al., Science 273 (1996), 1386-1389).

Part of the DNA component of the RNA-DNA oligonucleotide used for thispurpose is homologous to a nucleic acid sequence of an endogenousprotein, but exhibits a mutation in comparison with the nucleic acidsequence of the endogenous protein or comprises a heterologous regionenclosed by the homologous regions.

Base pairing of the homologous regions of the RNA-DNA oligonucleotideand of the endogenous nucleic acid molecule followed by homologousrecombination allows the mutation or heterologous region contained inthe DNA component of the RNA-DNA oligonucleotide to be transferred intothe genome of a plant cell. This leads to a reduced activity of theprotein involved in the starch metabolism.

As an alternative, the enzyme activities which are involved in thestarch metabolism can be reduced in the plant cells by a cosuppressioneffect. This method is known to the skilled worker and is described, forexample, by Jorgensen (Trends Biotechnol. 8 (1990), 340-344), Niebel etal., (Curr. Top. Microbiol. Immunol. 197 (1995), 91-103), Flavell et al.(Curr. Top. Microbiol. Immunol. 197 (1995), 43-46), Palaqui andVaucheret (Plant. Mol. Biol. 29 (1995), 149-159), Vaucheret et al.,(Mol. Gen. Genet. 248 (1995), 311-317), de Bome et al. (Mol. Gen. Genet.243 (1994), 613-621) and other sources.

To inhibit the synthesis, in the transformed plants, of a plurality ofenzymes involved in starch biosynthesis, it is possible to use DNAmolecules for transformation which simultaneously contain, in antisenseorientation and under the control of a suitable promoter, a plurality ofregions which encode the relevant enzymes. Each sequence may be underthe control of its own promoter, or, alternatively, the sequences can betranscribed by a joint promoter as a fusion, so that synthesis of theproteins in question is inhibited to approximately the same or to adifferent extent. As regards the length of the individual coding regionswhich are used in such a construct, what has already been said above forthe generation of antisense constructs also applies here. In principle,there is no upper limit for the number of antisense fragmentstranscribed starting from a promoter in such a DNA molecule. However,the resulting transcript should not, as a rule, exceed a length of 25kb, preferably 15 kb.

The nucleic acid molecules according to the invention make it possibleto transform plant cells and simultaneously to inhibit the synthesis ofa plurality of enzymes.

Moreover, it is possible to introduce the nucleic acid moleculesaccording to the invention into traditional mutants which are deficientor defective with regard to one or more starch biosynthesis genes(Shannon and Garwood, 1984, in Whistler, BeMiller and Paschall, Starch:Chemistry and Technology, Academic Press, London, 2nd Edition: 25-86).These defects can relate, for example, to the following proteins:granule-bound (GBSS I) and soluble starch synthases (SSS I, I, III andIV), branching enzymes (BE I, IIa and IIb), debranching enzymes(R-enzymes, isoamylases, pullulanases), disproportioning enzymes andplastid starch phosphorylases.

The present invention thus also relates to transgenic plant cellsobtainable by a process according to the invention which have beentransformed with a nucleic acid molecule or vector according to theinvention, and to transgenic plant cells derived from cells transformedin this way. The cells according to the invention contain a nucleic acidmolecule according to the invention, this preferably being linked toregulatory DNA elements which ensure transcription in plant cells, inparticular to a promoter. The cells according to the invention can bedistinguished from naturally occurring plant cells, inter alia, by thefact that they contain a nucleic acid molecule according to theinvention which does not occur naturally in these cells, or by the factthat such a molecule exists integrated at a location in the cell'sgenome where it does not occur otherwise, i.e. in a different genomicenvironment. Furthermore, the transgenic plant cells according to theinvention can be distinguished from naturally occurring plant cells bythe fact that they contain at least one copy of a nucleic acid moleculeaccording to the invention stably integrated into their genome, ifappropriate in addition to copies of such a molecule which occurnaturally in the cells. If the nucleic acid molecule(s) introduced intothe cells is (are) additional copies to molecules which already occurnaturally in the cells, then the plant cells according to the inventioncan be distinguished from naturally occurring plant cells in particularby the fact that this (these) additional copy (copies) is (are)localized at sites of the genome at which it (they) do(es) not occurnaturally. This can be checked, for example, with the aid of a Southernblot analysis.

Preferred plant cells according to the invention are those in which theenzyme activity of individual enzymes which are involved in starchmetabolism is increased or reduced by at least 10%, especiallypreferably by at least 30%, and very especially preferably by at least50%.

Moreover, the plant cells according to the invention can bedistinguished from naturally occurring plant cells preferably by atleast one of the following features: if the nucleic acid moleculeaccording to the invention which has been introduced is heterologousrelative to the plant cell, the transgenic plant cells exhibittranscripts of the nucleic acid molecules according to the inventionwhich have been introduced. This can be detected by, for example,northern blot analysis. For example, the plant cells according to theinvention contain one or more proteins encoded by a nucleic acidmolecule according to the invention which has been introduced. This canbe detected by, for example, immunological methods, in particular bywestern blot analysis.

If the nucleic acid molecule according to the invention which has beenintroduced is homologous relative to the plant cell, the cells accordingto the invention can be distinguished from naturally occurring cells,for example, on the basis of the additional expression of nucleic acidmolecules according to the invention. For example, the transgenic plantcells contain more or fewer transcripts of the nucleic acid moleculesaccording to the invention. This can be detected by, for example,northern blot analysis. “More” or “fewer” in this context meanspreferably at least 10% more or fewer, preferably at least 20% more orfewer and especially preferably at least 50% more or fewer transcriptsthan corresponding untransformed cells. Furthermore, the cellspreferably exhibit a corresponding (At least 10%, 20% or 50%,respectively) increase or decrease in the content of protein accordingto the invention. The transgenic plant cells can be regenerated intointact plants by techniques known to the skilled worker.

The plants obtainable by regenerating the transgenic plant cellsaccording to the invention, and processes for the generation oftransgenic plants by regenerating intact plants from the plant cellsaccording to the invention, are also subject matter of the presentinvention. Another subject matter of the invention are plants whichcontain the transgenic plant cells according to the invention. Inprinciple, the transgenic plants can be plants of any species, i.e. notonly monocotyledonous, but also dicotyledonous plants. The plants arepreferably useful plants, i.e. plants which are grown by man for thepurposes of nutrition or for technical, in particular industrial,purposes. They are preferably starch-storing plants such as, forexample, cereal species (rye, barley, oats, maize, wheat, sorghum andmillet, sago etc.), rice, peas, marrowfat peas, cassava, potatoes,tomatoes, oilseed rape, soybeans, hemp, flax, sunflowers, cowpeas, mungbeans or arrowroot.

The invention also relates to propagation material of the plantsaccording to the invention, for example fruits, seeds, tubers, rootstocks, seedlings, cuttings, calli, protoplasts, cell cultures etc.

Altering the enzymatc activities of the enzymes involved in starchmetabolism results in the synthesis, in the plants generated by theprocess according to the invention, of a starch with a modifiedstructure.

A large number of cloning vectors are available for preparing theintroduction of foreign genes into higher plants, vectors which containa replication signal for E. coli and a marker gene for the selection oftransformed bacterial cells. Examples of such vectors are pBR322, pUCseries, M13mp series, pACYC184 and the like. The desired sequence can beintroduced into the vector at a suitable restriction cleavage site. Theresulting plasmid is used for transforming E. coli cells. Transformed E.coli cells are cultured in a suitable medium and then harvested andlysed. The plasmid is recovered. The analytical methods forcharacterizing the plasmid DNA obtained are generally restrictionanalyses, gel electrophoreses and other methods of biochemistry andmolecular biology (Sambrook et al. loc. cit.). After each manipulation,the plasmid DNA can be cleaved and DNA fragments obtained linked toother DNA sequences. Each plasmid DNA sequence can be cloned into thesame or other plasmids.

A large number of techniques are available for introducing DNA into aplant host cell. These techniques encompass the transformation of plantcells with T-DNA using Agrobacterium tumefaciens or Agrobacteriumrhizogenes as the means for transformation, protoplast fusion by meansof polyethylene glycol (PEG), injection, DNA electroporation, theintroduction of DNA by means of the biolistic method, and otherpossibilities.

The injection and electroporation of DNA into plant cells requires noparticular aspects of the plasmids or the DNA used per se. Simpleplasmids such as, for example, pUC derivatives can be used. However, ifintact plants are to be regenerated from such transformed cells, thepresence of a selectable marker gene is required.

Depending on the method of introducing desired genes into the plantcell, further DNA sequences may be required. If, for example, the Ti orRi plasmid is used for transforming the plant cell, at least the rightborder, but frequently the right and left border, of the Ti and Riplasmid T-DNA must be linked to the genes to be introduced as flankingregion. If agrobacteria are used for the transformation, the DNA to beintroduced must be cloned into specific plasmids, either into anintermediate vector or into a binary vector. The intermediate vectorscan be integrated into the agrobacterial Ti or Ri plasmid by homologousrecombination owing to sequences which are homologous to sequences inthe T-DNA. The former also contains the vir region required for T-DNAtransfer. Intermediate vectors cannot replicate in agrobacteria. Theintermediate vector can be transferred to Agrobacterium tumefaciens bymeans of a helper plasmid (conjugation). Binary vectors are capable ofreplication in E. coli and in agrobacteria. They contain a selectionmarker gene and a linker or polylinker which are framed by the left andright T-DNA border region. They can be transformed directly into theagrobacteria (Holsters et at. (1978) Mol. Gen. Genet. 163: 181-187). Theagrobacterium which acts as the host cell should contain a plasmidcarrying a vir region. The vir region is required for transferring theT-DNA into the plant cell. Additional T-DNA may be present. Theagrobacterium transformed in this way is used for transforming plantcells.

The use of T-DNA for transforming plant cells has been researchedintensively and been described in EP 120516; Hoekema, in: The BinaryPlant Vector System Offsetdrukkerij Kanters B.V., Alblasserdam (1985),Chapter V; Fraley et al., Crit. Rev. Plant. Sci., 4: 1-46 and An et al.(1985) EMBO J. 4: 277-287.

To transfer the DNA into the plant cell, plant explants can expedientlybe cocultured with Agrobacterium tumefaciens or Agrobacteriumrhizogenes. Intact plants can then be regenerated from the infectedplant material (for example leaf sections, stem segments, roots, butalso protoplasts, or plant cells which have been grown in suspensionculture) in a suitable medium which can contain antibiotics or biocidesfor selecting transformed cells. The resulting plants can then beexamined for the presence of the DNA which has been introduced. Otherpossibilities of introducing foreign DNA using the biolistic method orby protoplast transformation are known (cf., for example, Willmitzer, L,1993 Transgenic plants. In: Biotechnology, A Multi-Volume ComprehensiveTreatise (H. J. Rehm, G. Reed, A. Pühler, P. Stadler, eds.), Vol. 2,627-659, VCH Weinheim-New York-Basle-Cambridge).

While the transformation of dicotyledonous plants via Ti-plasmid vectorsystems with the aid of Agrobacterium tumefaciens is well established,more recent work suggests that even monocotyledonous plants are indeedaccessible to transformation by means of agrobacterium-based vectors(Chan et al., Plant Mol. Biol. 22 (1993), 491-506; Hiei et al., Plant J.6 (1994), 271-282).

Alternative systems for the transformation of monocotyledonous plantsare the transformation by means of the biolistic approach, protoplasttransformation, the electroporation of partially permeabilized cells,and the introduction of DNA by means of glass fibers.

Specifically, different methods have been described in the literaturefor the transformation of maize (cf., for example, WO 95/06128, EP 0 513849; EP 0 465 875). EP 292 435 describes a method with the aid of whichfertile plants can be obtained starting from a mucilage-free, friable,granular maize callus. In this context, Shillito et al. (Bio/Technology7 (1989), 581) have observed that the capacity of regenerating fertileplants requires starting from callus suspension cultures from which adividing protoplast culture with the capacity of regenerating plants canbe made. Following an in-vitro culture period of 7 to 8 months, Shillitoet al. obtained plants with viable progeny which, however, haveabnormalities with regard to morphology and reproductivity. Prioli andSöndahl (Bio/Technology 7 (1989), 589) also describe the regenerationand obtaining of fertile maize plants from maize protoplasts.

Once the DNA which has been introduced is integrated into the genome ofthe plant cell, it is, as a rule, stable therein and is also retained inthe progeny of the originally transformed cell. It normally contains aselection marker which imparts to the transformed plant cells resistanceto a biocide or an antibiotic such as kanamycin, G 418, bleomycin,hygromycin or phosphinotricin and the like. The individual marker chosenshould therefore allow selection of transformed cells over cells whichlack the DNA which has been introduced.

Within the plant, the transformed cells grow in the customary manner(see also McCormick et al.(1986) Plant Cell Reports 5:81-84). Theresulting plants can be grown normally and hybridized with plants whichhave the same transformed germ plasm or other germ plasm. The resultinghybrids have the corresponding phenotypic features.

Two or more generations should be grown to ensure that the phenotypicfeature is stably retained and inherited. Also, seeds should beharvested to ensure that the phenotype in question or other featureshave been retained.

Yet another subject matter of the invention is a process for theproduction of starch in a manner known per se, in which plant cellsaccording to the invention, plants according to the invention, plantparts according to the invention or propagation material according tothe invention are processed or integrated into the process.

Processes for extracting starch from plants or from starch-storing partsof plants are known to the skilled worker. Processes for extractingstarch from maize kernels are described, for example, by Eckhoff et al.(Cereal Chem. 73 (1996) 54-57). As a rule, the extraction of maizestarch on the industrial scale is performed by wet milling. Moreover,processes for extracting the starch from various starch-storing plantsare described, for example, in “Starch: Chemistry and Technology (eds:Whistler, BeMiller and Paschall (1994), 2nd Edition, Academic Press Inc.London Ltd; ISBN 0-12-746270-8; see, for example, Chapter XII, pages412-468: maize and sorghum starches: production; by Watson: ChapterXIII, pages 469-479: tapioca, arrowroot and sago starches: production;by Corbishley and Miller; Chapter XIV, pages 479-490: potato starch:production and uses; by Mitch; Chapter XV, pages 491 to 506: wheatstarch: production, modification and uses; by Knight and Oson; andChapter XVI, pages 507 to 528: rice starch: production and uses; byRohmer and Klem). Devices normally used in processes for extractingstarch from plant material are separators, decanters, hydrocyclones,spray dryers and fluidized-bed dryers.

Owing to the expression of a nucleic acid molecule according to theinvention, the transgenic plant cells and plants according to theinvention synthesize a starch which is modified in comparison to thestarch synthesized in wild-type plants for example with regard to itsphysico-chemical properties.

Yet another subject matter of the invention is the starch which can beobtained from a plant cell according to the invention, plant accordingto the invention, their propagation material or a method according tothe invention.

A further embodiment of the present invention also includes the use ofthe starch according to the invention in industry for the production offoodstuffs, packaging materials or disposable products.

The starch according to the invention can be modified by processes knownto the skilled worker and is suitable, in its unmodified or modifiedform, for a variety of applications in the food or non-food sector.

In principle, the possible uses of the starch according to the inventioncan be divided into two important sectors. One sector encompasses thehydrolisates of the starch, mainly glucose and glucose units, which areobtained by enzymatic or chemical methods. They are used as startingmaterial for other chemical modifications and processes such asfermentation. What may be important here is the simplicity andinexpensive design of a hydrolytic process as is currently performedessentially enzymatically using amyloglucosidase. What would be feasibleis a financial saving by using less enzyme. This could be caused byaltering the structure of the starch, for example increasing the surfacearea of the granule, by better degradability owing to a lower degree ofbranching, or by a steric structure which limits the accessibility forthe enzymes employed.

The other sector in which starch according to the invention can be usedas so-called native starch, due to its polymeric structure, can bedivided into two further fields of application:

1. The Food Industry

Starch is a traditional additive to a large number of foodstuffs inwhich its function is essentially to bind aqueous additives or to causeincreased viscosity or else increased gelling. Important characteristicsare the visco-elasticity, the sorptive characteristics, the swellingtemperature, the gelatinization temperature, the viscosity andthickening power, starch solubility, transparency and gel structure,thermal stability, shear stability, stability to acids, the tendency toundergo retrogradation, the film-forming capacity, the freeze-thawstability, digestibility and the ability of forming complexes with, forexample, inorganic or organic ions.

2. The Non-food Industry

In this important sector, starch is employed as an auxiliary for variouspreparation processes or as an additive in industrial products. Whenusing starch as an auxiliary, mention must be made, in particular, ofthe paper and board industry. Starch acts mainly for retardationpurposes (retaining solids), binding filler particles and fines, as astiffener and for dehydration. Moreover, the advantageous properties ofstarch regarding stiffness, strength, sound, touch, luster, smoothness,bonding strength and the surfaces are made use of.

2.1. The Paper and Board Industry

Within the papermaking process, four fields of application must bedistinguished, i.e. surface, coating, stock and spraying. With 80% ofthe consumption, surface starch accounts for by far the greatest starchquantity, 8% are used as coating starch, 7% as stock starch and 5% asspraying starch.

The demands on starch with regard to surface treatment are essentiallyhigh whiteness, an adapted viscosity, highly stable viscosity, good filmformation and low dust formation. When used for coating, the solidscontent, an adapted viscosity, a high binding capacity and a highpigment affinity play an important role. Of importance when used as anadditive to the stock is rapid, uniform, loss-free distribution, highmechanical strength and complete retention in the paper web. If thestarch is used in the spraying sector, again, an adapted solids content,high viscosity and a high binding capacity are of importance.

2.2. The Adhesives Industry

An important field of application for starches is in the adhesivesindustry, where the potential uses can be divided into four subsections:the use as a pure starch paste, the use in starch pastes which have beentreated with specialty chemicals, the use of starch as additive tosynthetic resins and polymer dispersions, and the use of starches asextenders for synthetic adhesives. 90% of the starch based adhesives isemployed in the sectors production of corrugated board, production ofpaper sacks and bags, production of composite materials for paper andaluminum, production of box board and gumming adhesives for envelopes,stamps and the like.

2.3. The Textiles and Textile Care Products Industry

An important field of application for starches as auxiliaries andadditive is the sector production of textiles and textile care products.The following four fields of application must be distinguished withinthe textiles industry: the use of starch as sizing agent, i.e. asauxiliary for smoothing and strengthening the burring behavior as aprotection from the tensile forces applied during weaving, and forincreasing resistance to abrasion during weaving, starch as a textilefinishing agent, in particular after quality-reducing pretreatments suchas bleaching, dyeing and the like, starch as thickener in thepreparation of dye pastes for preventing bleeding, and starch asadditive to chaining agents for sewing threads.

2.4. The Construction Materials Industry

The fourth field of application is the use of starches as additive inconstruction materials. An example is the production of gypsumplasterboards, where the starch which is admixed to the gypsum slurrygelatinizes with the water, diffuses to the surface of the plaster core,where it binds the boards to the core. Other fields of application arethe admixture to rendering and mineral fibers. In the case ofready-mixed concrete, starch products are employed for delaying binding.

2.5. Soil Stabilization

A limited market for starch products is the production of soilstabilizers, which are employed for the temporary protection of the soilparticles from water when the soil is disturbed artificially. Accordingto present knowledge, product combinations of starch and polymeremulsions equal the previously employed products with regard to theirerosion- and crust-reducing effect, but are markedly less expensive.

2.6. Use in Crop Protection Products and Fertilizers

One field of application for using starch is in crop protection productsfor altering the specific properties of the products. Thus, starches areemployed for improving the wettability of crop protection products andfertilizers, for the controlled release of the active ingredients, forconverting liquid active ingredients, volatile active ingredients and/oractive ingredients with an offensive odor into microcrystalline, stable,shapeable substances, for mixing incompatible compounds, and forextending the duration of action by reducing decomposition.

2.7. Pharmaceuticals, Medicine, and the Cosmetics Industry

Another field of application is the sector of pharmaceuticals, medicineand the cosmetics industry. In the pharmaceuticals industry, starchesare employed as binders for tablets or for diluting the binder incapsules. Moreover, starches are used as tablet disintegrants, sincethey absorb fluids after swallowing and swell within a short time tosuch an extent that the active ingredient is liberated. Medicinallubricating powders and wound powders are starch-based for reasons ofquality. In the cosmetics sector, starches are employed, for example, ascarriers of powder additives such as fragrances and salicylic acid. Arelatively large field of application for starch is toothpaste.

2.8. Addition of Starch to Coal and Briquettes

A field of application for starch is as additive to coal and briquettes.With an addition of starch, coal can be agglomerated, or briquetted, interms of high quantity, thus preventing early decomposition of thebriquettes. In the case of barbecue coal, the starch addition amounts tobetween 4 and 6%, in the case of calorized coal to between 0.1 and 0.5%.Moreover, starches are gaining importance as binders since the emissionof noxious substances can be markedly reduced when starches are added tocoal and briquettes.

2.9. Ore Slick and Coal Silt Processing

Furthermore, starch can be employed as flocculant in ore slick and coalsilt processing.

2.10. Foundry Auxiliary

A further field of application is as additive to foundry auxiliaries.Various casting processes require cores made with sands treated withbinders. The binder which is predominantly employed nowadays isbentonite, which is treated with modified starches, in most casesswellable starches.

The purpose of adding starch is to increase flowability and to improvethe binding power. In addition, the swellable starches can meet thedemands of production engineering, such as being cold-water dispersible,rehydratable and readily miscible with sand and having high waterbinding capacity.

2.11. Use in the Rubber Industry

In the rubber industry, starch is employed for improving the technicaland visual quality. The reasons are the improvement of the surfaceluster, the improvement of handle and of appearance, and to this endstarch is scattered over the tacky gummed surfaces of rubber materialsprior to cold curing, and also the improvement of the rubber'sprintability.

2.12. Production of Leather Substitutes

Modified starches may furthermore also be sold for the production ofleather substitutes.

2.13. Starch in Synthetic Polymers

In the polymer sector, the following fields of application can beenvisaged: the incorporation of starch degradation products in theprocessing process (starch is only a filler, there is no direct bondbetween the synthetic polymer and the starch) or, alternatively, theincorporation of starch degradation products in the production ofpolymers (starch and polymer form a stable bond).

The use of starch as pure filler is not competitive in comparison withother substances such as talc. However, this is different when thespecific properties of starch make an impact and thus markedly alter thespectrum of characteristics of the end products. An example of this isthe use of starch products in the processing of thermoplasts, such aspolyethylene. Here, the starch and the synthetic polymer are combined bycoexpression in the ratio 1:1 to give a master batch, from which variousproducts are produced together with granulated polyethylene, usingconventional process techniques. By incorporating starch in polyethylenefilms, an increased substance permeability in the case of hollow bodies,an improved permeability for water vapor, an improved antistaticbehavior, an improved antiblock behavior and an improved printabilitywith aqueous inks can be achieved. The current disadvantages relate tothe insufficient transparency, the reduced tensile strength, and areduced elasticity.

Another possibility is the use of starch in polyurethane foams. Byadapting the starch derivatives and by processing-engineeringoptimization, it is possible to control the reaction between syntheticpolymers and the starches' hydroxyl groups in a direct manner. Thisresults in polyurethane films which have the following spectrum ofproperties, owing to the use of starch: a reduced heat extensioncoefficient, a reduced shrinking behavior, an improved pressure-tensionbehavior, an increase in permeability for water vapor without alteringthe uptake of water, a reduced flammability and a reduced ultimatetensile strength, no drop formation of combustible parts, freedom fromhalogens, and reduced aging. Disadvantages which still exist are areduced printability and a reduced impact strength. Product developmentis currently no longer restricted to films. Solid polymer products suchas pots, slabs and dishes with a starch content of over 50% may also beproduced. Moreover, starch/polymer mixtures are considered advantageoussince their biodegradability is much higher.

Starch graft polymers have become exceedingly important owing to theirextremely high water binding capacity. They are products with a starchbackbone and a side chain of a synthetic monomer, grafted on using theprinciple of the free-radical chain mechanism. The starch graft polymerswhich are currently available are distinguished by a better binding andretention capacity of up to 1000 g water per g of starch, combined withhigh viscosity. The fields of application for these superabsorbers haveextended greatly in recent years and are, in the hygiene sector, theproducts diapers and pads, and, in the agricultural sector, for examplein seed coatings.

What is decisive for the application of novel, genetically modifiedstarches are, on the one hand, structure, water content, proteincontent, lipid content, fiber content, ash/phosphate content,amylose/amylopectin ratio, molecular mass distribution, the degree ofbranching, granule size, granule shape and crystallization, and, on theother hand, also the characteristics which affect the followingfeatures: viscoelasticity, sorption characteristics, gelatinizationtemperature, viscosity, thickening powder, solubility, gel structure,transparency, thermal stability, shear stability, stability to acids,tendency to undergo retrogradation, gel formation, freeze-thawstability, complex formation, iodine binding, film formation, adhesivepower, enzyme stability, digestibility and reactivity.

The production of modified starches by recombinant methods can, on theone hand, alter the properties, for example of the starch derived fromthe plant, in such a way that other modifications by means of chemicalor physical alterations are no longer required. On the other hand,starches which have been modified by recombinant methods may besubjected to further chemical modifications, which leads to furtherimprovements in quality for some of the above-described fields ofapplication. These chemical modifications are known in principle. Theyare, in particular, modifications by thermal and pressure treatment,treatment with organic or inorganic acids, enzymatic treatment,oxidations or esterifications, which lead, for example, to the formationof phosphate starches, nitrate starches, sulfate starches, xanthatestarches, acetate starches and citrate starches. Moreover, mono- orpolyhydric alcohols in the presence of strong acids may be employed forproducing starch ethers, resulting in starch alkyl-ethers, O-allylethers, hydroxyalkyl ethers, O-carboxy/methyl ethers, N-containingstarch ethers, P-containing starch ethers, S-containing starch ethers,crosslinked starches or starch graft polymers.

A use of the starches according to the invention is in industrialapplication, preferably for foodstuffs or the production of packagingmaterials and dispersible articles.

The examples which follow serve to illustrate the invention andconstitute in no way a restriction.

Abbreviations used:

BE branching enzyme bp base pair IPTG isopropylβ-D-thiogalactopyranoside SS soluble starch synthase PMSFphenylmethylsulfonyl fluoride

Media and solutions used in the examples:

20 × SSC 175.3 g NaCl 88.2 g sodium citrate to 1000 ml withtwice-distilled H₂O pH 7.0 with 10N NaOH Buffer A 50 mM Tris-HCl pH 8.02.5 mM DTT 2 mM EDTA 0.4 mM PMSF 10% glycerol 0.1% sodium dithioniteBuffer B 50 mM Tris-HCl pH 7.6 2.5 mM DTT 2 mM EDTA Buffer C 0.5M sodiumcitrate pH 7.6 50 mM Tris-HCl pH 7.6 2.5 mM DTT 2 mM EDTA 10 × TBS 0.2MTris-HCl pH 7.5 5.0M NaCl 10 × TBST 10 × TBS 0.1% (v/v) Tween 20 Elutionbuffer 25 mM Tris pH 8.3 250 mM glycine Dialysis buffer 50 mM Tris-HClpH 7.0 50 mM NaCl 2 mM EDTA 14.7 mM β-mercaptoethanol 0.5 mM PMSFProtein buffer 50 mM sodium phosphate buffer pH 7.2 10 mM EDTA 0.5 mMPMSF 14.7 mM β-mercaptoethanol

DESCRIPTION OF THE FIGURES

FIG. 1 represents a schematic RVA temperature profile (viscosity vs.time [min]) with the viscosimetric parameters T=gelatinizationtemperature, temperature at the point in time when gelatinizationstarts; Max specifies the maximum viscosity; Min specifies the minimumviscosity; Fin specifies the viscosity at the end of the measurement;Set is the difference (Δ) of Min and Fin (setback).

The following methods were used in the examples

1. Cloning Method

The vector pBluescript II SK (Stratagene) was used for cloning into E.coli.

For the transformation of plants, the gene constructs were cloned intothe binary vector pBinAR Hyg (Höfgen & Willmitzer, 1990, Plant Sci.66:221-230) and pBinB33-Hyg.

2. Bacterial Strains and Plasmids

The E. coli strain DH5α (Bethesda Research Laboratories, Gaithersburgh,USA) was used for the Bluescript vector p Bluescript II KS (Stratagene)and for the pBinAR Hyg and pBinB33 Hyg constructs. The E. coli strainXL1-Blue was used for the in vivo exclusion.

pBinAR

The plasmid pBinAR is a derivative of the binary vector plasmid pBin19(Bevan, 1984, Nucl. Acid Res. 12:8711-8721), which was constructed asfollows: a 529 bp fragment encompassing the nucleotides 6909-7437 of thecauliflower mosaic virus promoter 35S promoter was isolated from plasmidpDH51 as EcoRI/KpnI fragment (Pietrzak et al., 1986), ligated betweenthe EcoRI and KpnI cleavage sites of the pUC18 polylinker, and wastermed plasmid pUC18-35S. With the aid of the restriction endonucleasesHindIII and PvuII, a 192 bp fragment was isolated from plasmid pAGV40(Herrera-Estrella et al., 1983), which encompasses DNA of the Ti-plasmidpTiACH5 (Gielen et al, 1984, EMBO J.:835-846) (nucleotides 11749-11939).After the PvuII cleavage sites had been provided with SphI linkers, thefragment was ligated between the SpHI and HindIII cleavage sites ofpUC18-35S, and this was termed plasmid pA7. Moreover, the entirepolylinker comprising the 35S promoter and the ocs terminator wasexcised with EcoRI and HindIII and ligated into the appropriatelycleaved pBin19. This gave rise to the plant expression vector pBinAR(Höfgen and Willmitzer, 1990).

pBinB33

The promoter of the Solanum tuberosum patatin gene B33 (Rocha-Sosa etal., 1989) was ligated, as DraI fragment (nucleotides −1512-+14) intothe vector pUC19, which had been cleaved with Sst I and which had beenmade blunt-ended with the aid of T4-DNA polymerase. This gave rise toplasmid pUC19-B33. The B33 promoter was excised from this plasmid withEcoRI and SmaI and ligated into the appropriately cleaved vector pBinAR.This gave rise to the plant expression vector pBinB33.

pBinAR-Hyg

Starting from plasmid pA7 (cf. description of vector pBinAR), theEcoRI-HindIII fragment comprising the 35S promoter, the ocs terminatorand the portion of the polylinker situated between the 35S promoter andthe ocs terminator was introduced into the appropriately cleaved plasmidpBin-Hyg.

pBinB33-Hyg

Starting from plasmid pBinB33, the EcoRI-HindIII fragment comprising theB33 promoter, part of the polylinker and the ocs terminator was excisedand ligated into the appropriate cleaved vector pBin-Hyg. This gave riseto the plant expression vector pBinB33-Hyg.

3. Transformation of Agrobacterium tumefaciens

The DNA was transferred by direct transformation using the method ofHöfgen&Willmitzer (1988, Nucleic Acids Res. 16:9877). The plasmid DNA oftransformed agrobacteria was isolated using the method of Bimboim&Doly(1979, Nucleic Acids Res. 7:1513-1523), subjected to suitablerestriction cleavage, and then analyzed by gel electrophoresis.

4. Transformation of Potatoes

The transformation of the plasmids into the potato plants (Solanumtuberosum L.cv. Desiree, Vereinigte Saatzuchten eG, Ebstorf) was carriedout with the aid of the Agrobacterium tumefaciens strain C58C1 (Dietzeet al. (1995) in Gene Transfer to Plants. pp. 24-29, eds.: Potrykus, I.and Spangenberg, G., Springer Verlag, Deblaere et al., 1985, Nucl. AcidsRes. 13:4777-4788).

Ten small leaves of a sterile potato culture which had been scarifiedwith a scalpel were placed into 10 ml of MS medium (Murashige&Skoog(1962) Physiol. Plant. 15: 473) supplemented with 2% sucrose andcontaining 50 ml of an Agrobacterium tumefaciens overnight culture grownunder selection conditions. After the culture had been shaken gently for3-5 minutes, it was incubated for 2 more days in the dark. For callusinduction, the leaves were then placed on MS medium supplemented with1.6% glucose, 5 mg/l naphthylacetic acid, 0.2 mg/l benzylaminopurin, 250mg/l claforan, 50 mg/l kanamycin, and 0.80% Bacto agar. After the leaveshad been incubated for one week at 25° C. and 3000 Lux, they were placedon MS medium supplemented with 1.6% glucose, 1.4 mg/l zeatin ribose, 20mg/l naphthylacetic acid, 20 mg/l gibberellic acid, 250 mg/l claforan,50 mg/l kanamycin and 0.80% Bacto agar, to induce shoots.

5. Plant Culture Regime

Potato plants were kept in the greenhouse under the followingconditions:

light period 16 h at 25,000 Lux and 22° C. dark period 8 h at 15° C.atmospheric humidity 60%

6. Radiolabeling of DNA Fragments

The DNA fragments were radiolabeled with the aid of a DNA Random PrimerLabeling Kit by Boehringer Mannheim (Germany) following themanufacturers instructions.

7. Determination of Starch Synthase Activity

Determination of starch synthase activity was done by determining theincorporation of ¹⁴C glucose from ADP[¹⁴C glucose] into a product whichis insoluble in methanol/KCl, as described by Denyer & Smith, 1992,Planta 186:609-617.

8. Detection of Soluble Starch Synthases in the Native Gel

To detect the activity of soluble starch syntheses by non-denaturing gelelectrophoresis, tissue samples of potato tubers were hydrolyzed in 50mM Tris-HCl pH 7.6, 2 mM DTT, 2.5 mM EDTA, 10% glycerol and 0.4 mM PMSF.The electrophoresis was carried out in a MiniProtean II chamber(BioRAD). The monomer concentration of the gels, which had a thicknessof 1.5 mm, was 7.5% (w/v), and 25 mM Tris-glycine pH 8.4 was used as gelbuffer and running buffer. Identical amounts of protein extract wereapplied and separated for 2 hours at 10 mA per gel.

The activity gels were subsequently incubated in 50 mM Tricine-NaOH pH8.5, 25 mM potassium acetate, 2 mM EDTA, 2 mM DTT, 1 mM ADP-glucose,0.1% (w/v) amylopectin and 0.5 M sodium citrate. The glucans formed werestained with Lugol's solution.

9. Starch Analysis

The starch formed by the transgenic potato plants was characterized bythe following methods:

a) Determination of the Amylose/amylopectin Ratio in Starch from PotatoPlants

Starch was isolated from potato plants by standard methods, and theamylose:amylopectin ratio was determined by the method described byHovenkamp-Hermelink et al. (Potato Research 31 (1988) 241-246).

b) Determination of the Phosphate Content

In potato starch, some glucose units may be phosphorylated on the carbonatoms of position C2, C3 and C6. To determine the degree ofphosphorylation at position C6 of the glucose, 100 mg of starch werehydrolyzed for 4 hours at 95° C. in 1 ml of 0.7 M HCl (Nielsen et al.(1994) Plant Physiol. 105: 111-117). Following neutralization with 0.7 MKOH, 50 ml of the hydrolysate were subjected to a visual-enzymatic testto determine glucose-6-phosphate. The change in absorption of the testbatch (100 mM imidazole/HCl; 10 mM MgCl₂; 0.4 mM NAD; 2 unitsLeuconostoc mesteroides glucose-6-phosphate dehydrogenase; 30° C.) wasmonitored at 334 nm.

The total phosphate was determined as described by Ames, 1996, Methodsin Enzymology VIII, 115-118.

c) Analysis of the Amylopectin Side Chains

To analyze distribution and length of the side chains in the starchsamples, 1 ml of a 0.1% starch solution was digested with 0.4 U ofisoamylase (Megazyme International Ireland Ltd., Bray, Ireland)overnight at 37° C. in 100 mM sodium citrate buffer, pH 3.5.

The rest of the analysis was carried out as described by von Tomlinsonet al., (1997), Plant J. 11:31-47, unless otherwise specified.

d) Granule Size Determination

The granule size was determined using a “Lumosed” photosedimentometer byRetsch GmbH, Germany. To this end, 0.2 g of starch was suspended inapprox. 150 ml of water and immediately measured. The program suppliedby the manufacturer calculated the mean diameter of the starch granules,assuming an average starch density of 1.5 g/l.

e) Gelatinization Properties

The gelatinization or viscosity properties of the starch were recordedusing a Viscograph E by Brabender OHG, Germany, or a Rapid ViscoAnalyser, Newport Scientific Pty Ltd., Investment Support Group,Warriewood NSW 2102, Australia. When using the Viscograph E, asuspension of 30 g of starch in 450 ml of water was subjected to thefollowing heating program: heat from 50° C. to 96° C. at 3°/min, holdfor 30 minutes, cool to 30° C. at 3°/min and hold again for 30 minutes.The temperature profile gave characteristic gelatinization properties.

When measuring using the Rapid Visco Analysers (RVA) a suspension of 2 gof starch in 25 ml of water was subjected to the following heatingprogram: suspend for 60 seconds at 50° C., heat from 50° C. to 95° C. at12°/min, hold for 2.5 minutes, cool to 50° C. at 12° C./min and holdagain for 2 minutes. The RVA temperature profile gave the viscosimetricparameters of the tested starches for the maximum viscosity (Max), theend viscosity (Fin), the gelatinization temperature (T), the minimumviscosity (Min) occurring after the maximum viscosity and the differencebetween minimum viscosity and end viscosity (Setback, Set) (cf. Table 1and FIG. 1).

f) Determination of the Gel Strength

To determine the gel strength by means of a Texture Analyser, 2 g ofstarch were gelatinized in 25 ml of water (cf. RVA measurement) and thenstored for 24 hours in a sealed container at 25° C. with the exclusionof air. The samples were mounted underneath the probe (circular stamp)of a TA-XT2 Texture Analyser (Stable Micro Systems), and the gelstrength was determined with the following parameter settings:

Test speed 0.5 mm Penetration depth 7 mm Contact area (of the stamp) 113mm² Pressure/contact area 2 g

10. Determination of Glucose, Fructose and Sucrose

To determine the glucose, fructose and sucrose content, small tuberportions (diameter approx. 10 mm) of potato tubers were frozen in liquidnitrogen and then extracted for 30 minutes at 80° C. in 0.5 ml of 10 mMHEPES, pH 7.5; 80% (vol/vol) ethanol. The supernatant, which containsthe solubles, was removed and the volume was determined. The supernatantwas used for determining the amount of soluble sugars. The quantitativedetermination of soluble glucose, fructose and sucrose was carried outin a batch of the following composition

100.0 mM imidazole/HCl, pH 6.9 1.5 mM MgCl₂ 0.5 mM NADP⁺ 1.3 mM ATP10-50 μl sample 1.0 U yeast glucose-6-phosphate dehydrogenase

The batch was incubated for 5 minutes at room temperature. The sugarswere subsequently determined photometrically by measuring the absorptionat 340 nm after the successive addition of 1.0 unit yeast hexokinase (todetermine glucose), 1.0 unit yeast phosphoglucoisomerase (to determinefructose), and 1.0 unit yeast invertase (to determine sucrose).

USE EXAMPLES Example 1 Isolation of a cDNA Fragment Encoding Potatoα-glucosidase

Total RNA of potato tuber tissue directly underneath (approx. 1 cm)germinating shoots were prepared by standard methods (Sambrook et al.,1989).

The purified total RNA was used as starting material for the preparationof poly A+ RNA (Oligotex, mRNA Purification Kit, in accordance with themanufacturer's instructions). 5 μg of this poly A+ RNA were used togenerate a cDNA library (λ ZAPII, Stratagene).

Approximately 3×10⁵ plaque-forming units (pfus) of this unamplified cDNAlibrary (primary library) were plated following the manufacturer'sinstructions (Stratagene) for plaque lifting. The radiolabeled probe(Random Primed DNA Labeling Kit, following the manufacturer'sinstructions) used for plaque hybridization was the sequence of GenbankAccession No. T76451. The filters were prehybridized for 4 hours at 42°C. (buffer: 5×SSC, 0.5% BSA, 5×Denhardt, 1% SDS, 40 mM phosphate buffer,pH 7.2, 100 mg/l herring sperm DNA, 25% formamide) and subsequentlyhybridized for 14 hours at the same temperature. After hybridization,the filters were washed 3× for 20 minutes with 3×SSC, 0.5% SDS at 42° C.and autoradiographed. Hybridizing plaques were singled out, and thephages isolated were used for in-vivo excision following themanufacturer's instructions. Plasmid DNA from the bacterial coloniesobtained were isolated, employed for sequence analysis and identified asSEQ ID NO: 1.

A plasmid DNA isolated in this manner was deposited on Jul. 24, 1998 atthe Deutsche Sammlung für Mikroorganismen und Zellkulturen (DSMZ)Brunswick, FRG, under the number DSM 12347.

Example 2 Preparation of Plasmid p35SαSSI-Hyg

A 1831 bp Asp718/XbaI fragment containing a partial cDNA encoding thepotato SSS I (Abel, G., (1995) PhD Thesis, Free University of Berlin),was inserted between the Asp 718 and XbaI cleavage site of the vectorpBinAR-Hyg in antisense orientation relative to the 35S promoter.

Example 3 Preparation of Plasmid p35S-SSI-Kan

A 2384 bp EcoRI fragment containing a cDNA encoding potato SSI (Abel1995, loc. cit.) was made blunt-ended and introduced into the vectorpBinAR, which had previously been cut with SmaI, in sense orientationrelative to the 35S promoter.

Example 4 Preparation of Plasmid p35SαSSII-Kan

A 1959 bp SmaI/Asp718 fragment containing a partial cDNA encoding thepotato SS II (Abel, 1995, termed GBSS II therein) was made blunt-endedand introduced into the SmaI cleavage site of the vector pBinAR inanti-sense orientation relative to the 35S promoter.

Example 5 Preparation of Plasmid pB33-SSII-Hyg

A 2619 bp SmaI/SalI fragment containing a cDNA encoding the potato SS II(Abel, 1995, loc. cit.) was introduced into the vector pBinB33-Hyg,which had previously been cut with SmaI and SalI in sense orientationrelative to the B33 promoter.

Example 6 Preparation of Plasmid p35SαSSIII-Hyg

A 4212 bp Asp718/XbaI fragment containing a cDNA encoding the potato SSIII (Abel et al 1996, Plant J. 10(6):981-991), was inserted between theAsp718 and the XbaI cleavage site of the vector pBinAR-Hyg in antisenseorientation relative to the 35S promoter.

Example 7 Preparation of Plasmid p35S-SSIII-Kan

A 4191 bp EcoRI fragment containing a cDNA encoding potato SS III (Abelet al., 1996, loc. cit.), was made blunt-ended and introduced into theSmaI cleavage site of the vector pBinAR in sense orientation relative tothe 35S promoter.

Example 8 Preparation of Plasmid pB33αBEαSSIII-Kan

A 1650 bp HindII fragment which contains a partial cDNA encoding thepotato BE enzyme (Kossmann et al., 1991, Mol. & Gen. Genetics230(1-2):3944) was made blunt-ended and introduced in antisenseorientation relative to the B33 promoter into the vector pBinB33 whichhad been precut with SmaI. The resulting plasmid was cut open withBamHI. A 1362 Bp BamHI fragment containing a partial cDNA encoding thepotato SS III enzyme (Abel et al., 1996, loc. cit.) was introduced intothe cleavage site, again in antisense orientation relative to the B33promoter.

Example 9 Preparation of Plasmid p35SαSSII-αSSIII-Kan

A 1546 bp EcoRV/HincII fragment containing a partial cDNA encoding thepotato SS II (Abel, 1995, loc. cit.) was cloned into the vectorpBluescript II KS which can been cut with EcoRV/HincII, then excisedagain by digestion with Asp718/BamHI and introduced in antisenseorientation relative to the 35S promoter into the vector pBinAR whichhad been digested in the same manner. Then, a 1356 bp BamHI fragmentcontaining a partial cDNA encoding the potato SS III (Abel et al., 1996,loc. cit.), was introduced into the BamHI cleavage site of the vectorpBinAR-αSSII, again in antisense orientation.

Example 10 Preparation of Plasmid pB33αSSIαSSIαSSIII-Kan

A 2384 bp EcoRI fragment containing a cDNA encoding the potato SS I(Abel, 1995, loc. cit.) was made blunt-ended and cloned into the SmaIcleavage site of the pBinB33 vector in antisense orientation relative tothe B33 promoter. A 1362 bp BamHI fragment containing a partial cDNAencoding the potato SS III (Abel et al., 1996, loc. cit.) was introducedinto the BamHI cleavage site of the resulting vector, again in antisenseorientation relative to the B33 promoter.

Example 11 Preparation of Plasmid p35SαSSII-Hyg

A 1959 bp SmaI/Asp718 fragment containing a partial cDNA encoding the SSII (Abel, 1995, loc. cit.), was made blunt-ended and introduced into theSmaI cleavage site of the pBinAR-Hyg vector in antisense orientationrelative to the 355 promoter.

Example 12 Introduction of the Plasmids into the Genome of Potato Cells

The plasmids stated in Examples 1 to 11 were transferred, eitherindividually and/or in succession, into agrobacteria, with the aid ofwhich potato cells were transformed as described above. Subsequently,intact plants were regenerated from the transformed plant cells.

Example 13 Characterization of the Physicochemical Properties of theModified Starches

As a result of the transformation, the transgenic potato plants showed achange in the physico-chemical properties of the starches synthesized bythem. The starch formed by these plants differs for example from starchsynthesized in wild-type plants with regard to its phosphate or amylosecontent, the viscosity or gelatinization properties determined by RVA,and its chromotographic behavior.

2 1 2272 DNA Solanum tuberosum CDS (135)..(2180) coding sequence ofalpha-glucosidase 1 cgaatacgaa taaccgacgc taaccatcaa cgatgggaagtgccggaaga aattctccac 60 cgtccaccac cgccgtcgcc gccgtcaacc tccaactcctcatcagaaaa ccactcccca 120 attaccctct ctaa ccc aaa ctc aga cct aga gttcac cct tca caa cac 170 Pro Lys Leu Arg Pro Arg Val His Pro Ser Gln His1 5 10 cat ccc att cag ctt cac cgt ccg ccg gcg ctc cac cgg gga tac tct218 His Pro Ile Gln Leu His Arg Pro Pro Ala Leu His Arg Gly Tyr Ser 1520 25 ttt cga tac ttc gcc gga gtt agt cat ggg gtt ttg ctt ctg agt agc266 Phe Arg Tyr Phe Ala Gly Val Ser His Gly Val Leu Leu Leu Ser Ser 3035 40 aat ggc atg gat att gtg tat acg ggt gat agg att agt tac aag gtg314 Asn Gly Met Asp Ile Val Tyr Thr Gly Asp Arg Ile Ser Tyr Lys Val 4550 55 60 att gga ggg tta att gat ttg tat ttc ttt gcc gga cct tcg ccg gaa362 Ile Gly Gly Leu Ile Asp Leu Tyr Phe Phe Ala Gly Pro Ser Pro Glu 6570 75 atg gtg gtg gat cag tat act cag ctt att ggt cgt cct gct gct atg410 Met Val Val Asp Gln Tyr Thr Gln Leu Ile Gly Arg Pro Ala Ala Met 8085 90 cca tat tgg tct ttc gga ttt cac caa tgc cgg tgg ggt tac aag aat458 Pro Tyr Trp Ser Phe Gly Phe His Gln Cys Arg Trp Gly Tyr Lys Asn 95100 105 att gat gat gtt gaa ctg gta gtg gat agt tat gca aag tct aga ata506 Ile Asp Asp Val Glu Leu Val Val Asp Ser Tyr Ala Lys Ser Arg Ile 110115 120 ccg ctg gag gtt atg tgg act gat att gat tac atg gat ggt ttt aag554 Pro Leu Glu Val Met Trp Thr Asp Ile Asp Tyr Met Asp Gly Phe Lys 125130 135 140 gac ttc aca ctc gat cca gtt aac ttc cca ctg gag cgg gta attttt 602 Asp Phe Thr Leu Asp Pro Val Asn Phe Pro Leu Glu Arg Val Ile Phe145 150 155 ttt ctc agg aag ctt cat cag aat gat cag aaa tat gta cta atagta 650 Phe Leu Arg Lys Leu His Gln Asn Asp Gln Lys Tyr Val Leu Ile Val160 165 170 gat cca gga att agc atc aac aat aca tat gac acc tat agg agaggc 698 Asp Pro Gly Ile Ser Ile Asn Asn Thr Tyr Asp Thr Tyr Arg Arg Gly175 180 185 atg gaa gca gat gtc ttc ata aaa cgc gat aat atg ccc tac caaggg 746 Met Glu Ala Asp Val Phe Ile Lys Arg Asp Asn Met Pro Tyr Gln Gly190 195 200 gtt gtt tgg cca ggg aat gtt tat tat cct gat ttt cta aat ccagct 794 Val Val Trp Pro Gly Asn Val Tyr Tyr Pro Asp Phe Leu Asn Pro Ala205 210 215 220 act gaa gta ttt tgg aga aat gaa att gag aag ttc cag gatctc gta 842 Thr Glu Val Phe Trp Arg Asn Glu Ile Glu Lys Phe Gln Asp LeuVal 225 230 235 cct ttt gat ggc ctg tgg ctt gac atg aat gaa ttg tca aacttc ata 890 Pro Phe Asp Gly Leu Trp Leu Asp Met Asn Glu Leu Ser Asn PheIle 240 245 250 act tcc cct cct aca cca tca tct acc ttt gat gat cct ccctac aag 938 Thr Ser Pro Pro Thr Pro Ser Ser Thr Phe Asp Asp Pro Pro TyrLys 255 260 265 ata aac aac tct ggc gat cac ttg ccc atc aat tat aga acagtt cca 986 Ile Asn Asn Ser Gly Asp His Leu Pro Ile Asn Tyr Arg Thr ValPro 270 275 280 gcc act tct aca cat ttt ggt gat aca atg gag tat aat gtccat aac 1034 Ala Thr Ser Thr His Phe Gly Asp Thr Met Glu Tyr Asn Val HisAsn 285 290 295 300 ctt tat gga tta ctt gaa tct aga gcc act tat agt gcattg gtt aat 1082 Leu Tyr Gly Leu Leu Glu Ser Arg Ala Thr Tyr Ser Ala LeuVal Asn 305 310 315 gtc act ggt aaa agg cca ttc att ctt gta aga tca actttt ctt ggc 1130 Val Thr Gly Lys Arg Pro Phe Ile Leu Val Arg Ser Thr PheLeu Gly 320 325 330 tct ggc aga tac acg tca cat tgg act gga gat aat gctgct acc tgg 1178 Ser Gly Arg Tyr Thr Ser His Trp Thr Gly Asp Asn Ala AlaThr Trp 335 340 345 aac gat ttg gca tac tcc att cct act atc ttg agc tttgga ttg ttt 1226 Asn Asp Leu Ala Tyr Ser Ile Pro Thr Ile Leu Ser Phe GlyLeu Phe 350 355 360 gga att cca atg gtt gga gct gat ata tgt ggt ttt tcaagt aac act 1274 Gly Ile Pro Met Val Gly Ala Asp Ile Cys Gly Phe Ser SerAsn Thr 365 370 375 380 act gaa gag ctt tgc cgc cgc tgg att cag ctt ggagca ttc tat cca 1322 Thr Glu Glu Leu Cys Arg Arg Trp Ile Gln Leu Gly AlaPhe Tyr Pro 385 390 395 ttt gca aga gac cac tct gct aag gac aca acc ccccaa gag ctc tat 1370 Phe Ala Arg Asp His Ser Ala Lys Asp Thr Thr Pro GlnGlu Leu Tyr 400 405 410 agt tgg gat tca gtt gct gca gca gcc aag aaa gtcctt ggg ctc cga 1418 Ser Trp Asp Ser Val Ala Ala Ala Ala Lys Lys Val LeuGly Leu Arg 415 420 425 tat cag tta ctt cca tac ttt tat atg ctt atg tacgag gca cat ata 1466 Tyr Gln Leu Leu Pro Tyr Phe Tyr Met Leu Met Tyr GluAla His Ile 430 435 440 aaa ggg act ccc att gca cga ccc ctc ttc ttc tctttc cct caa gat 1514 Lys Gly Thr Pro Ile Ala Arg Pro Leu Phe Phe Ser PhePro Gln Asp 445 450 455 460 gcc aag aca ttt gat atc agc aca cag ttc cttctc ggt aaa ggt gtc 1562 Ala Lys Thr Phe Asp Ile Ser Thr Gln Phe Leu LeuGly Lys Gly Val 465 470 475 atg atc tca cct ata ctt aag caa gga gca acctct gtt gat gca tat 1610 Met Ile Ser Pro Ile Leu Lys Gln Gly Ala Thr SerVal Asp Ala Tyr 480 485 490 ttc cct gct gga aac tgg ttt gac ctc ttc aattac tct cgc tct gtg 1658 Phe Pro Ala Gly Asn Trp Phe Asp Leu Phe Asn TyrSer Arg Ser Val 495 500 505 agt ttg aat caa gga aca tat atg aca ctt gacgca cca cca gat cat 1706 Ser Leu Asn Gln Gly Thr Tyr Met Thr Leu Asp AlaPro Pro Asp His 510 515 520 ata aat gta cat gtt cgt gaa ggg aac ata ttggtc atg caa ggg gaa 1754 Ile Asn Val His Val Arg Glu Gly Asn Ile Leu ValMet Gln Gly Glu 525 530 535 540 gca atg aca aca caa gct gct cag agg actgca ttc aaa ctc ctt gtc 1802 Ala Met Thr Thr Gln Ala Ala Gln Arg Thr AlaPhe Lys Leu Leu Val 545 550 555 gtg ctg agc agc agc aaa aac agc aca ggagaa cta ttt gtg gac gat 1850 Val Leu Ser Ser Ser Lys Asn Ser Thr Gly GluLeu Phe Val Asp Asp 560 565 570 gac gat gag gtg cag atg gga aga gag ggaggg agg tgg acg cta gtt 1898 Asp Asp Glu Val Gln Met Gly Arg Glu Gly GlyArg Trp Thr Leu Val 575 580 585 aag ttt aac agc aat atc att ggc aat aaaatt gtg gtt aaa tca gag 1946 Lys Phe Asn Ser Asn Ile Ile Gly Asn Lys IleVal Val Lys Ser Glu 590 595 600 gtt gtg aat gga cga tat gcg ctg gat caagga ttg gtc ctt gaa aag 1994 Val Val Asn Gly Arg Tyr Ala Leu Asp Gln GlyLeu Val Leu Glu Lys 605 610 615 620 gtg aca tta ttg gga ttt gaa aat gtgaga gga ttg aag agc tat gag 2042 Val Thr Leu Leu Gly Phe Glu Asn Val ArgGly Leu Lys Ser Tyr Glu 625 630 635 ctt gtt gga tca cac cag caa ggg aacaca aca atg aag gaa agt ctt 2090 Leu Val Gly Ser His Gln Gln Gly Asn ThrThr Met Lys Glu Ser Leu 640 645 650 aag cag agt gga cag ttt gtt act atggaa atc tca ggg atg tca ata 2138 Lys Gln Ser Gly Gln Phe Val Thr Met GluIle Ser Gly Met Ser Ile 655 660 665 ttg ata ggg aaa gag ttc aaa ttg gagcta tac atc att act 2180 Leu Ile Gly Lys Glu Phe Lys Leu Glu Leu Tyr IleIle Thr 670 675 680 taacaaatga attaagttat atacgcttgt tgtatgaaattttctttcat ttatcaatgc 2240 agtttaattt atgataaaaa aaaaaaaaaa aa 2272 2682 PRT Solanum tuberosum 2 Pro Lys Leu Arg Pro Arg Val His Pro Ser GlnHis His Pro Ile Gln 1 5 10 15 Leu His Arg Pro Pro Ala Leu His Arg GlyTyr Ser Phe Arg Tyr Phe 20 25 30 Ala Gly Val Ser His Gly Val Leu Leu LeuSer Ser Asn Gly Met Asp 35 40 45 Ile Val Tyr Thr Gly Asp Arg Ile Ser TyrLys Val Ile Gly Gly Leu 50 55 60 Ile Asp Leu Tyr Phe Phe Ala Gly Pro SerPro Glu Met Val Val Asp 65 70 75 80 Gln Tyr Thr Gln Leu Ile Gly Arg ProAla Ala Met Pro Tyr Trp Ser 85 90 95 Phe Gly Phe His Gln Cys Arg Trp GlyTyr Lys Asn Ile Asp Asp Val 100 105 110 Glu Leu Val Val Asp Ser Tyr AlaLys Ser Arg Ile Pro Leu Glu Val 115 120 125 Met Trp Thr Asp Ile Asp TyrMet Asp Gly Phe Lys Asp Phe Thr Leu 130 135 140 Asp Pro Val Asn Phe ProLeu Glu Arg Val Ile Phe Phe Leu Arg Lys 145 150 155 160 Leu His Gln AsnAsp Gln Lys Tyr Val Leu Ile Val Asp Pro Gly Ile 165 170 175 Ser Ile AsnAsn Thr Tyr Asp Thr Tyr Arg Arg Gly Met Glu Ala Asp 180 185 190 Val PheIle Lys Arg Asp Asn Met Pro Tyr Gln Gly Val Val Trp Pro 195 200 205 GlyAsn Val Tyr Tyr Pro Asp Phe Leu Asn Pro Ala Thr Glu Val Phe 210 215 220Trp Arg Asn Glu Ile Glu Lys Phe Gln Asp Leu Val Pro Phe Asp Gly 225 230235 240 Leu Trp Leu Asp Met Asn Glu Leu Ser Asn Phe Ile Thr Ser Pro Pro245 250 255 Thr Pro Ser Ser Thr Phe Asp Asp Pro Pro Tyr Lys Ile Asn AsnSer 260 265 270 Gly Asp His Leu Pro Ile Asn Tyr Arg Thr Val Pro Ala ThrSer Thr 275 280 285 His Phe Gly Asp Thr Met Glu Tyr Asn Val His Asn LeuTyr Gly Leu 290 295 300 Leu Glu Ser Arg Ala Thr Tyr Ser Ala Leu Val AsnVal Thr Gly Lys 305 310 315 320 Arg Pro Phe Ile Leu Val Arg Ser Thr PheLeu Gly Ser Gly Arg Tyr 325 330 335 Thr Ser His Trp Thr Gly Asp Asn AlaAla Thr Trp Asn Asp Leu Ala 340 345 350 Tyr Ser Ile Pro Thr Ile Leu SerPhe Gly Leu Phe Gly Ile Pro Met 355 360 365 Val Gly Ala Asp Ile Cys GlyPhe Ser Ser Asn Thr Thr Glu Glu Leu 370 375 380 Cys Arg Arg Trp Ile GlnLeu Gly Ala Phe Tyr Pro Phe Ala Arg Asp 385 390 395 400 His Ser Ala LysAsp Thr Thr Pro Gln Glu Leu Tyr Ser Trp Asp Ser 405 410 415 Val Ala AlaAla Ala Lys Lys Val Leu Gly Leu Arg Tyr Gln Leu Leu 420 425 430 Pro TyrPhe Tyr Met Leu Met Tyr Glu Ala His Ile Lys Gly Thr Pro 435 440 445 IleAla Arg Pro Leu Phe Phe Ser Phe Pro Gln Asp Ala Lys Thr Phe 450 455 460Asp Ile Ser Thr Gln Phe Leu Leu Gly Lys Gly Val Met Ile Ser Pro 465 470475 480 Ile Leu Lys Gln Gly Ala Thr Ser Val Asp Ala Tyr Phe Pro Ala Gly485 490 495 Asn Trp Phe Asp Leu Phe Asn Tyr Ser Arg Ser Val Ser Leu AsnGln 500 505 510 Gly Thr Tyr Met Thr Leu Asp Ala Pro Pro Asp His Ile AsnVal His 515 520 525 Val Arg Glu Gly Asn Ile Leu Val Met Gln Gly Glu AlaMet Thr Thr 530 535 540 Gln Ala Ala Gln Arg Thr Ala Phe Lys Leu Leu ValVal Leu Ser Ser 545 550 555 560 Ser Lys Asn Ser Thr Gly Glu Leu Phe ValAsp Asp Asp Asp Glu Val 565 570 575 Gln Met Gly Arg Glu Gly Gly Arg TrpThr Leu Val Lys Phe Asn Ser 580 585 590 Asn Ile Ile Gly Asn Lys Ile ValVal Lys Ser Glu Val Val Asn Gly 595 600 605 Arg Tyr Ala Leu Asp Gln GlyLeu Val Leu Glu Lys Val Thr Leu Leu 610 615 620 Gly Phe Glu Asn Val ArgGly Leu Lys Ser Tyr Glu Leu Val Gly Ser 625 630 635 640 His Gln Gln GlyAsn Thr Thr Met Lys Glu Ser Leu Lys Gln Ser Gly 645 650 655 Gln Phe ValThr Met Glu Ile Ser Gly Met Ser Ile Leu Ile Gly Lys 660 665 670 Glu PheLys Leu Glu Leu Tyr Ile Ile Thr 675 680

I claim:
 1. An isolated nucleic acid molecule encoding a protein withthe function of a potato α-glucosidase, selected from the groupconsisting of a) nucleic acid molecules which encode a protein whichcomprises the amino acid sequence stated under SEQ ID NO: 2, b) nucleicacid molecules which comprise the nucleotide sequence shown under SEQ IDNO: 1; c) nucleic acid molecules which have over 85% sequence identityto the nucleotide sequence shown under SEQ ID NO:1, and d) nucleic acidmolecules whose nucleotide sequence deviates from the sequence of thenucleic acid molecules stated under b) owing to the degeneracy of thegenetic code.
 2. The nucleic acid molecule as claimed in claim 1, whichis a deoxyribonucleic acid molecule.
 3. The nucleic acid molecule asclaimed in claim 1, which is a cDNA molecule.
 4. The nucleic acidmolecule as claimed in claim 1, which is a ribonucleic acid molecule. 5.An isolated nucleic acid molecule which specifically hybridizes with thenucleic acid molecule as claimed in claim 1, under highly stringentconditions followed by a wash step, wherein hybridization temperature isat 68° C. and is followed by a wash at 68° C. in a wash buffercontaining 0.2×SSC.
 6. A vector comprising the nucleic acid molecule asclaimed in claim
 1. 7. A vector comprising the nucleic acid molecule asclaimed in claim 1, wherein the nucleotide sequence encoding a proteinwith the function of an α-glucosidase or parts thereof is present insense or antisense orientation.
 8. A vector comprising the nucleic acidmolecule as claimed in claim 1, which is linked to regulatory elementsthat initiate transcription of RNA in a cell.
 9. A host cell which istransformed with the nucleic acid molecule as claimed in claim 1, orwith a the vector as claimed in claim 6; or a cell which is derived fromthe host cell, and which comprises said nucleic acid molecule or vector.10. A method for making a transgenic plant cell which synthesizes amodified starch, wherein the nucleic acid molecule as claimed in claim1, or the vector as claimed in claim 6, is integrated into the genome ofa plant cell.
 11. A plant cell which is made by the method of claim 10.12. A transgenic plant comprising the nucleic acid molecule of claim 1.13. A transgenic plant comprising the plant cell of claim 11.